Nucleic acid molecules, polypeptides, antibodies and compositions for treating and detecting influenza virus infection

ABSTRACT

Polynucleotides and polypeptides which participate in influenza virus infection of cells and nucleic acid molecules, which include a polynucleotide sequence capable of specifically binding the polypeptides of the present invention. Also provided are methods of using such nucleic acid molecules, polynucleotides and antibodies directed thereagainst for diagnosing, treating and preventing influenza virus infection.

RELATED PATENT APPLICATION

This application is a Divisional of U.S. patent application Ser. No.10/546,034 filed Aug. 18, 2005, which is a National Phase Application ofPCT/IL2004/000182 having International Filing Date of Feb. 24, 2004,which claims the benefit of priority of U.S. Provisional Application No.60/449,863 filed Feb. 27, 2003. The contents of the above applicationare all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to nucleic acid molecules, polypeptides,antibodies and pharmaceutical composition containing same, which can beutilized for treating and detecting influenza virus infection invertebrates such as avian, swines and humans.

Influenza viruses have been a major cause of mortality and morbidity inman throughout recorded history. Influenza epidemics occur at regularintervals, which vary widely in severity but which always causesignificant mortality and morbidity, most frequently in the elderlypopulation. An influenza infection produces an acute set of symptomsincluding headache, cough, fever and general malaise. In severe cases orsituations involving pre-existing pulmonary or cardiovascular disease,hospitalization is required. Pneumonia due to direct viral infection ordue to secondary bacterial or viral invasion is the most frequentcomplication. For a review on the clinical aspects of influenza virusinfection see Douglas (1990) New England Journal of Medicine,322:443-450.

Influenza viruses are currently divided into three types: A, B. and C,based upon differences in internal antigenic proteins; while the A and Btypes are closely related and account for most infections, the type Cinfluenza virus represents a distant third in disease-causing potentialand is probably of little public health concern. Although overall genehomology is less than 30%, between the A and B types, these virusesshare a common ancestor and include eight RNAs of negative sensepolarity. Hemagglutinin (HA) and neuraminidase (NA) are expressed on thesurface of the lipid containing virus particles and are primarilyresponsible for the antigenic changes observed in influenza viruses.

New strains of influenza caused by antigenic drift appear at regularfrequency, usually annually, and begin a cycle of infection, whichtravels around the globe. Little is known about how individual epidemicsare initiated. Major new subtypes of influenza appear less frequentlybut can result in major pandemics.

It will be appreciated that up to 20% of the population may developinfluenza infection in any given year and influenza epidemics areresponsible for 20,000 deaths per year in the U.S. [Palese (2002) J.Clin. Invest. 110:9-13]. By far the most catastrophic impact ofinfluenza during the past 100 years was the pandemic of 1918, is whichcost more than 500,000 lives in the U.S. and lowered life expectancy byalmost 10 years [Heilman (1990) Clin. North Am. 37:669-688].

Given the impact of influenza on individuals and on society thechallenge at present is to generate highly potent prophylactic toolswhich can be used to prevent influenza infection in subjects which areat considerable risk of infection such as young children and the elderlypopulation.

Several approaches have been undertaken to uncover novel anti influenzaagents.

Inactivated influenza virus vaccines—The most effective way to deal withthe influenza virus for a population at risk of severe complications isby prevention. To be effective, current vaccines must contain an A, Band preferably C virus components. To prepare vaccines, the viralstrains are grown in embryonated eggs, and the virus is then purifiedand made noninfectious by chemical inactivation. Use of the availableinfluenza vaccine is an effective way to lower the mortality in apopulation, however due to the ever-changing nature of the virus, thedevelopment of a vaccine with the appropriate composition to protectagainst the currently circulating virus strains is complex andexpensive. Moreover, patient compliance in receiving the vaccine isgenerally very low. Thus, large numbers of patients at risk of seriouscomplications from influenza virus go unprotected.

Cold adapted influenza virus vaccines—The generation of temperaturesensitive influenza viruses as live vaccines has been attempted becausethe pathogenicity in animals and mammals is significantly attenuated[Wareing (2001) Vaccine 19:3320-3330; Maasab (1990) Adv. Biotechnol.Processes 14:203-242]. Typically, to generate cold adapted viruses theinfluenza viruses are passaged in chicken kidney cells and inembryonated eggs to adapt growth thereof at 25° C. Thus, the annuallyadapted vaccine formulations can be genetically engineered to includethe two genes which encode major viral surface antigens (i.e., HA andNA) reflecting the antigens found in current strains, whereas theremaining six genes derived from the cold-adapted master strains. Suchlive-virus vaccines can induce local neutralizing immunity andcell-mediated immune responses, which may be associated with a longerlasting and cross-reactive immunity than is elicited by chemicallyinactivated virus preparations. However, the use of live vaccinesrequires extensive monitoring against unexpected complications, whichmight arise from the spread of virulent revertants essentiallyexplaining the nonexistence of licensure for such therapy in the U.S.

Genetically engineered live influenza virus vaccines—The advent oftechniques for engineering site-specific changes in the genomes of RNAviruses rendered it possible to develop new vaccine approaches [Enami(1990) Proc. Natl. Acad. Sci. USA 87:3802-3805; Garcia-Sastre (1998)Trends. Biotechnol. 16:230-235]. Thus, generation of virus particleswhich undergo only a single cycle of replication has been demonstratedby Watanbe and co-workers [(2002), J. Virol. 76:767-773]. Infection ofcells with a preparation of virus particles lacking the NEP expressinggene (NS2) produces viral proteins but does not result in the formationof infectious particles. Thus, these preparations induce a protectiveantibody response and stimulate a strong cell-mediated immune responsewithout allowing the replication of infectious virus. Another approachfor virus attenuation is the generation of a replication defectivestrain which M2 gene is eliminated. Such a deletion mutant growsefficiently in tissue culture but only poorly in mice and thusrepresents a potential live virus vaccine candidate [Watanbe (2001) J.Virol. 75:5656-5662]. However, frequently the infectious titers of suchengineered viruses are too low to be useful in a clinical setting.

DNA vaccination—This approach involves the topical administration oradministration via injection of plasmid DNA encoding one or moreinfluenza proteins. However, to date reports on DNA vaccination againstinfluenza have been limited to studies in animal models and notherapeutic efficacy has been demonstrated in human subjects [Donnelly(1995) Nat. Med. 1:583-587; Ljungberg (2000) 268:244-25-; Kodihalli(2000) Vaccine 18:2592-2599].

Antiviral agents—Four antiviral agents are approved at present in theU.S.; amantidine and rimantidine are chemically related inhibitors ofthe ion channel M2 protein which is involved in viral uncoating [Hay(1985) EMBO J 4:3021-3024], and zanamivir and oseltamivir are NAinhibitors [Palese (1976) J. Gen. Virol. 33:159-63], preventing theproper release of influenza virus particles from the cytoplasmicmembrane. These antiviral drugs are important adjuncts for any medicalintervention against influenza, and may be used in prophylaxis againstthe virus (excluding zanamivir which has not yet been approved).Furthermore, these agents can be of significant value in case a newpandemic strain emerge against which a vaccine has not been developed.

Despite overall advantages, the widespread use of currently availableantiviral agents is limited by concerns over side effects, patientscompliance and the possible emergence of drug-resistant variants.

Antisense—Attempts at the inhibition of influenza virus using antisenseoligonucleotides have been reported. Leiter and co-workers have targetedphosphodiester and phosphorothioate oligonucleotides to influenza A andinfluenza C viruses. Leiter, J., Agrawal, S., Palese, P. & Zamecnik, P.C., Proc. Natl. Acad. Sci. USA, 87:3430-3434 (1990). In this studypolymerase PB1 gene and mRNA were targeted in the vRNA 3′ region andmRNA 5′ region, respectively. Sequence-specific inhibition of influenzaA was not observed although some specific inhibition of influenza C wasnoted. No other influenza virus segments or mRNAs were targeted.

There is thus a widely recognized need for, and it would be highlyadvantageous to have compositions, which can be used to diagnose andtreat influenza virus infection devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anucleic acid molecule comprising a polynucleotide sequence capable ofspecifically binding a polypeptide participating in influenza virusinfection of cells.

According to further features in preferred embodiments of the inventiondescribed below, the polynucleotide sequence is selected from the groupconsisting of SEQ ID Nos. 11 and 12.

According to still further features in the described preferredembodiments the polypeptide is an influenza virus polypeptide.

According to still further features in the described preferredembodiments the polynucleotide sequence is capable of binding a regionof hemagglutinin defined by amino acid coordinates 91-261 of SEQ ID NO:1.

According to still further features in the described preferredembodiments the polypeptide is a host cell polypeptide.

According to still further features in the described preferredembodiments the host cell polypeptide is a sialic acid receptor.

According to another aspect of the present invention there is provided amethod of generating a molecule capable of inhibiting influenza virusinfection, the method comprising: (a) contacting a plurality of nucleicacid molecules with a polypeptide participating in influenza virusinfection of cells; (b) identifying at least one nucleic acid moleculefrom the plurality of nucleic acid molecules capable of specificallybinding the polypeptide; and (c) isolating the at least one nucleic acidmolecule capable of binding the polypeptide, thereby generating themolecule capable of inhibiting influenza virus infection.

According to still further features in the described preferredembodiments the method further comprising generating the plurality ofnucleic acid molecules using a combinatorial synthesis approach prior to(a).

According to still further features in the described preferredembodiments the method further comprising modifying the plurality ofnucleic acid molecules prior to (a) or following (c).

According to still further features in the described preferredembodiments the method further comprising repeating steps (a) to (c).

According to yet another aspect of the present invention there isprovided a pharmaceutical composition comprising a nucleic acid moleculeincluding a polynucleotide sequence capable of specifically binding apolypeptide participating in influenza virus infection of cells and aphysiologically acceptable carrier.

According to still another aspect of the present invention there isprovided an article-of-manufacture comprising packaging material and apharmaceutical composition identified for treating or preventinginfluenza infection being contained within the packaging material, thepharmaceutical composition including, as an active ingredient, a nucleicacid molecule including a polynucleotide sequence capable ofspecifically binding a polypeptide participating in influenza virusinfection of cells.

According to an additional aspect of the present invention there isprovided a method of treating or preventing influenza virus infectioncomprising providing to a subject in need thereof, a therapeuticallyeffective amount of a nucleic acid molecule including a polynucleotidesequence capable of specifically binding a polypeptide participating ininfluenza virus infection of cells, thereby treating or preventing theinfluenza virus infection.

According to still further features in the described preferredembodiments the providing is effected by: (i) administering of thenucleic acid molecule; and/or (ii) administering a polynucleotideexpressing the nucleic acid molecule.

According to yet an additional aspect of the present invention there isprovided a method of identifying influenza virus in a biological sample,the method comprising: (a) contacting the biological sample with anucleic acid molecule including a polynucleotide sequence capable ofspecifically binding an influenza virus polypeptide; and (b) detectingthe nucleic acid molecule bound to the influenza virus polypeptide inthe biological sample, to thereby identify the influenza infection.

According to still an additional aspect of the present invention thereis provided a method of targeting an antiviral agent to an influenzavirus infected tissue, the method comprising administering to a subjectin need thereof a therapeutic effective amount of the antiviral agentconjugated to a nucleic acid molecule including a polynucleotidesequence capable of specifically binding an influenza virus polypeptide,thereby targeting the antiviral agent to the influenza infected tissue.

According to a further aspect of the present invention there is provideda composition of matter comprising an antiviral agent conjugated to anucleic acid molecule including a polynucleotide sequence capable ofspecifically binding a polypeptide participating in influenza virusinfection of cells.

According to still further features in the described preferredembodiments wherein the polypeptide is an influenza virus polypeptide.

According to still further features in the described preferredembodiments the polypeptide is selected from the group consisting ofhemagglutinin, neuraminidase, RNA-directed RNA polymerase core proteins,M1 matrix protein, M2 matrix protein and NS proteins.

According to still further features in the described preferredembodiments the polynucleotide sequence is capable of binding a regionof hemagglutinin defined by amino acid coordinates 91-261 of SEQ ID NO:1.

According to still further features in the described preferredembodiments the polypeptide is a host cell polypeptide.

According to still further features in the described preferredembodiments the host cell polypeptide is a sialic acid receptor.

According to still further features in the described preferredembodiments the polynucleotide sequence is single stranded.

According to still further features in the described preferredembodiments the polynucleotide sequence is DNA.

According to still further features in the described preferredembodiments the polynucleotide sequence is RNA.

According to still further features in the described preferredembodiments the nucleic acid molecule further comprising a detectablelabel.

According to still further features in the described preferredembodiments the polynucleotide sequence includes 2′-fluoro (2′-F)modified nucleotides.

According to still further features in the described preferredembodiments the polynucleotide sequence is selected having a lengthbetween 10 to 35 nucleotides.

According to still further features in the described preferredembodiments the pharmaceutical composition further includes an agent.

According to still further features in the described preferredembodiments agent is selected from the group consisting of animmunomodulatory agent, an antibiotic, an antiviral agent, an antisensemolecule and a rybosyme.

According to yet a further aspect of the present invention there isprovided a polypeptide useful for vaccination against influenza virus,the polypeptide comprising an amino acid sequence being at least 60%homologous to SEQ ID NO: 13 as determined using the BestFit software ofthe Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap creation penalty equals 8 and gapextension penalty equals 2, wherein the polypeptide does not include theHA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising a polypeptide includingan amino acid sequence being at least 60% homologous to SEQ ID NO: 13 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, the polypeptide notincluding the HA2 domain of influenza virus and a pharmaceuticallyacceptable carrier or diluent.

According to still a further aspect of the present invention there isprovided an antibody or antibody fragment comprising an antigen bindingsite specifically recognizing a polypeptide including an amino acidsequence being at least 60% homologous to SEQ ID NO: 13 as determinedusing the BestFit software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap creation penaltyequals 8 and gap extension penalty equals 2, wherein the polypeptidedoes not include the HA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided an isolated polynucleotide encoding a polypeptide including anamino acid sequence being at least 60% homologous to SEQ ID NO: 13 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, wherein thepolypeptide does not include the HA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided a nucleic acid construct comprising the isolated polynucleotideencoding a polypeptide including an amino acid sequence being at least60% homologous to SEQ ID NO: 13 as determined using the BestFit softwareof the Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap creation penalty equals 8 and gapextension penalty equals 2, wherein the polypeptide does not include theHA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided a host cell comprising the nucleic acid construct.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising a polynucleotideencoding a polypeptide including an amino acid sequence being at least60% homologous to SEQ ID NO: 13 as determined using the BestFit softwareof the Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap creation penalty equals 8 and gapextension penalty equals 2, the polypeptide not including the HA2 domainof influenza virus and a pharmaceutically acceptable carrier or diluent.

According to still a further aspect of the present invention there isprovided a method of treating or preventing influenza virus infectioncomprising providing to a subject in need thereof, a therapeuticallyeffective amount of a polypeptide including an amino acid sequence beingat least 60% homologous to SEQ ID NO: 13 as determined using the BestFitsoftware of the Wisconsin sequence analysis package, utilizing the Smithand Waterman algorithm, where gap creation penalty equals 8 and gapextension penalty equals 2, wherein the polypeptide does not include theHA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided a method of treating or preventing influenza virus infectioncomprising providing to a subject in need thereof, a therapeuticallyeffective amount of an antibody or antibody fragment including anantigen binding site specifically recognizing a polypeptide including anamino acid sequence being at least 60% homologous to SEQ ID NO: 13 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, wherein thepolypeptide does not include the HA2 domain of influenza virus.

According to still a further aspect of the present invention there isprovided a method of identifying influenza virus in a biological sample,the method comprising: (a) contacting the biological sample with anantibody or antibody fragment including an antigen binding sitespecifically recognizing a polypeptide including an amino acid sequencebeing at least 60% homologous to SEQ ID NO: 13 as determined using theBestFit software of the Wisconsin sequence analysis package, utilizingthe Smith and Waterman algorithm, where gap creation penalty equals 8and gap extension penalty equals 2, wherein the polypeptide does notinclude the HA2 domain of influenza virus; and (b) detectingimmunocomplexes including the antibody or antibody fragment in thebiological sample, to thereby identify the influenza virus in thebiological sample.

According to still further features in the described preferredembodiments the polypeptide is as set forth in SEQ ID NOs. 13-15.

According to still further features in the described preferredembodiments the amino acid sequence is as set forth in SEQ ID NOs.13-15.

According to still further features in the described preferredembodiments the amino acid sequence is defined by amino acid coordinates91-261 of SEQ ID NO: 1.

According to still further features in the described preferredembodiments the amino acid sequence is defined by amino acid coordinates116-261 of SEQ ID NO: 1.

According to still further features in the described preferredembodiments the amino acid sequence is defined by amino acid coordinates116-245 of SEQ ID NO: 1.

According to still further features in the described preferredembodiments the antibody or antibody fragment further includes a label.

According to still further features in the described preferredembodiments the detecting the immunocomplexes is effected by quantifyingintensity of the label following (b).

According to still a further aspect of the present invention there isprovided a nucleic acid molecule as set forth in SEQ ID NO: 11 or 12.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing nucleic acid molecules,polypeptides, antibodies generated thereagainst and compositionscontaining the same which can be used to diagnose and treat influenzavirus infection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-c are schematic illustrations adapted from Eaton (1997) Curr.Opin. Chem. Biol. 1:10-16 depicting nucleic acid modifications, whichcan be incorporated in the nucleic acid molecules of the presentinvention. FIG. 1 a shows 2′-deoxyuridines and uridines modified atposition 5. FIG. 1 b shows 2′-deoxyadenines, adenines and guanosinesmodified at position 8. FIG. 1 c shows 2′-modified uridines.

FIGS. 2 a-b are histograms depicting binding levels of influenzaspecific aptamers generated according to the teachings of the presentinvention (A21 and A22) and control single stranded aptamer to aninfluenza intact virus or the HA₉₁₋₂₆₁ peptide as determined by ELISA.Note a significant binding of A21 and A22 to the viral peptide ascompared to control nucleic acid is notable (p=0.042 and p=0.0008,respectively), and a significant reduction in A21 binding to the intactvirus as compared to the A22 aptamer (p=0.017).

FIGS. 2 c-e are schematic illustrations of proposed secondary structuresas generated by the DNAdraw software (18) of the A22 aptamer (FIG. 2 b),the A21 aptamer (FIG. 2 c) and control oligonucleotide coding for theNP147-158 (FIG. 2 d).

FIG. 3 a is a dose response curve showing the effect of A22 aptamer ofthe present invention on viability of influenza virus treated MDCK cellsas determined using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. Note, the highest protective effect was achieved using A22 at theconcentration range of 50 to 100 pmoles.

FIG. 3 b is a histogram presentation depicting the protective effects ofthe A21 and A22 aptamers (each at 50 pmols) of the present invention onH3N2 and H2N2 infected MDCK cells.

FIG. 3 c is a histogram presentation illustrating a cell protectiveeffect of A22 independent of the host cell proteins as determined by anMTT assay. MDCK cells were incubated with influenza virus 60 minutesfollowing treatment for the indicated time periods with 50 pmole of A22.Note the insignificant difference between treated and control groups(p=0.237 for 30 minutes and p=0.09 for 60 minutes). Likewise, nosignificant difference in cell viability was evident between the twoincubation times (p>0.05).

FIG. 3 d is a histogram illustrating the protective effect of A22 oninfected MDCK cells as determined by an MTT assay. MDCK cells wereincubated with influenza virus for 30 min or 60 min prior to treatmentwith 50 pmole A22 for 60 min. Note that although an insignificant effectof A22 on 60 minutes virus treated MDCK cells was evident, a significantprotective effect of A22 on 30 minutes virus treated MDCK cells was seenas compared to non-infected cells.

FIGS. 4 a-f are photomicrographs depicting the effect of A22 oninfection of cells with influenza. FIGS. 4 a-c are light microscopeimages of MDCK cells following infection with influenza (FIG. 4 a),following pre-treatment with A22 (FIG. 4 b) or non-infected MDCK cells.FIGS. 4 d-f are immunofluorescence images of MDCK cells following twodays of incubation with influenza virus (FIG. 4 d), influenza virus andA22 (FIG. 4 e), or A22 alone (FIG. 4 f).

FIGS. 5 a-f are photomicrographs showing lung sections of influenzavirus infected BALB/c mice in the presence or absence of A22. Mice inthe various treatment groups were sacrificed 6 days following intranasalinoculation with influenza virus and small portion of their lungs wereremoved and put into 10% neutralized formalin buffered for histologicalexaminations. Staining was effected with Haematoxylin and eosin. FIG. 5a—Lung section from a non-infected mouse; FIG. 5 b—Lung section frominfluenza virus infected mouse; FIG. 5 c—Lung section from a mouse ofgroup ‘−1 day ’, treated with A22 aptamer 1 day prior to viralinfection; FIG. 5 d—Mouse lung section of group ‘0 day’ treated with A22concomitantly with viral infection; FIGS. 5 e-f—are two differentsections from a lung of ‘+2 day’ group treated with A22 two daysfollowing the infection. It is estimated that about 60% of the lung ofthat mouse corresponded to the pattern in FIG. 5 f, which is similar tothe histology of the non-infected control, whereas 40% of the lungcontained bulk expansion of mononuclear cells (FIG. 5 e).

FIGS. 6 a-b are graphs illustrating the protective effect of A22 oninfluenza virus infected mice as determined by body weight (FIG. 6 a)and lung viral load (FIG. 6 b). Mice were infected with 100 HAU A/PortChalmers/1/73 by intranasal inoculation. −1 day group—mice treated with2.5 nmole A22 one day prior to viral inoculation. +2 day group—micetreated with 2.5 nmole A22 two day following viral inoculation. ‘0 daygroup—mice treated with A22 concomitantly with viral inoculation. Bodyweight of infected but untreated mice was compared to the weight of micetreated with A22 for the time intervals (FIG. 6 a). Alternatively,protection capacity of A22 was investigated by measuring viral load oflungs (FIG. 6 b).

FIG. 7 a is a graph depicting inhibition of mouse infection with severalstrains of influenza (each at 10 HAU) using the A22 aptamer.

FIG. 7 b is a graph depicting inhibition of mouse infection with theA/Texas/1/77 influenza strain using the A21 and A22 aptamers of thepresent invention, as well as control oligonucleotide and theanti-influenza drug Oseltamivir

FIGS. 8 a-d are graphs illustrating the cross-reactive effect ofantibodies against the recombinant HA₉₁₋₂₆₁ fragment with a varietyinfluenza virus strains as determined by ELISA. The IgG levels weremeasured by ELISA in serum samples of immunized (closed symbols) andnon-immunized (opened symbols) mice. FIG. 8 a—Port Chalmeras/1/73infected mice; FIG. 8 b—PR/8/34 infected mice; FIG. 8 c—Texas/1/77infected mice; FIG. 8 d—Japanese/57 infected mice; and FIG. 8 eillustrates strain-specific immune response induced by the intactA/Texas/1/77 (diamonds), A/Port Chalmers/1/73 (triangles)/ A/PR/8/34(circles) and A/Japanese /57 (crosses) viruses.

FIG. 9 a is a photomicrograph depicting an SDS-PAGE analysis of HA₉₁₋₁₀₈peptide purified by Ni-NTA column. M—molecular weight marker; 1-10 μg ofHA₉₁₋₁₀₈ peptide; 2-20 μg of HA₉₁₋₁₀₈ peptide.

FIG. 9 b is a graph depicting antigenisity of HA₉₁₋₁₀₈ peptide asdetermined by an ELISA assay. HA₉₁₋₂₆₁ peptide was coated to an ELISAplate and reacted with rabbit antiserum against HA₉₁₋₁₀₈ peptide (closedsquares) or against the intact A/Texas/77 influenza virus (opencircles). Control free antiserum is indicated by cross symbols.

FIGS. 10 a-b are graphic representations depicting the immunogenecity ofHA₉₁₋₂₆₁ peptide of the present invention as determined by an ELISAassay. Indicated sera dilutions from mice immunized with an HA₉₁₋₂₆₁peptide either intranasally (tiangles) or in the foot pad (diamonds) orwith a DNA vaccine (closed circles) corresponding to the peptide werecontacted with microtiter plates coated with the HA₉₁₋₂₆₁ peptide (FIG.10 a) or the intact virus (FIG. 10 b) and ELISA assay was effected. Serafrom mice immunized by DNA priming-protein boosting is indicated byclosed squares and from nonimmunized mice is indicated by cross symbols.Serum from mice immunized with control empty vector pCDNA3.1 isindicated with opened circles.

FIG. 10 c is a histogram depicting the cross reactivity of anti HA₉₁₋₂₆₁peptide with multiple influenza virus strains.

FIGS. 11 a-b are graphs illustrating the production of IgA antibodiesreactive with the HA₉₁₋₂₆₁ peptide of the present invention (FIG. 11 a)or A/Texas/77 virus (FIG. 11 b), following intranasal immunization ofmice with HA₉₁₋₂₆₁ peptide (closed triangles) or DNA vaccine (closedsquares) as determined by ELISA assay of lung homogenates. Combined DNApriming-protein fragment boosting is indicated by closed squares and nonimmunized control mice are indicated by cross symbols. Controlsimmunized with the vector pCDNA3.1 are indicated by opened squares.

FIGS. 12 a-b are histograms depicting proliferation of spleen cells frommice primed with HA₉₁₋₂₆₁ DNA and/or peptide in response to in vitrostimulation with HA₉₁₋₂₆₁ peptide (FIG. 12 a) or viral particles (FIG.12 b). The proliferation was monitored by thymidine uptake andrepresented as stimulation index compared to media control.

FIGS. 13 a-b are histograms depicting cytokine secretion by spleen cellsin response to influenza virus stimulation. Mice were immunized threetimes at 3-week intervals and retrieved spleen cells thereof werestimulated in vitro with inactivated influenza virus. Mean cytokineconcentrations quantitated by comparison with a standard curve ofpurified cytokines are presented.

FIGS. 14 a-b are histograms depicting CTL response in mice immunizedwith the peptide and/or DNA vaccines of the present invention. BALB/cmice were immunized with pHA₉₁₋₂₆₁ DNA or peptide and splenocytes wereassayed for virus-specific CTL activity. Data for each group is depictedby lysis of ⁵¹Cr labeled target cells at 20:1 (FIG. 14 a) and 50:1 (FIG.14 b) effector to target ratio.

FIGS. 15 a-b are histograms depicting protection against sublethalinfluenza virus challenge infection of mice immunized with pHA₉₁₋₂₆₁ DNAor peptide construct of the present invention. The mice were challenged4 weeks following the last immunization, and sacrificed 5 days later. A10⁻⁸ dilution of lung homogenate from each group was assayed for viruspresence by a haemagglutination assay. The results are presented aspercent virus positive lungs in each group at a 10⁻⁸ homogenatesdilution (FIG. 15 a), as well as LogEID₅₀ (FIG. 15 b). An asterikindicates a statistical significant difference (p<0.05).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of nucleic acid molecules, polynucleotides,polypeptides, antibodies, and pharmaceutical compositions, which can beused for treating and detecting influenza virus infection in vertebratessuch as avian, swines and humans.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Influenza is a major public health problem causing clinical morbidity,mortality and major economic losses each year of epidemic. To date,vaccination strategies, which constitute the basis of influenza control,have been directed at preventing morbidity and mortality in high-riskpopulation groups. However, due to the rapid and unpredictable change ofsurface proteins (i.e., hemagglutinin and neuraminidase) of theinfluenza virus, which lead to the emergence of new viral strains, thedevelopment of an effective vaccine is complex and expensive.

Currently available antiviral drugs include the viral M2 ion channelblockers amantidine and rimantadine and the neuraminidase blockerszanamivir (Relenza™) and oseltamivir (Tamiflu™), which prevent releaseand budding of the virus particles. While the M2 ion channel blockersare ineffective against the type B influenza virus which does not encodethe M2 protein and limited by severe side effects and acquiredresistance, the use of zanamivir is associated with airway resistanceand the use oseltamivir is highly priced.

As described hereinabove, the influenza virus encodes two surfaceantigens [neuraminidase and hemagglutinin (HA)], which undergo gradualchanges (i.e., antigenic shifts and drifts), leading to the highantigenic variations in influenza. The HA molecule (75-80 kD, GenBankAccession No. AF092062, SEQ ID NO: 1) is the most significant antigen indefining the serological specificity of the different virus strainsincluding a plurality of antigenic determinants, several of which are inregions that undergo sequence changes in different strains (i.e.,strain-specific determinants) and others in regions which are common tomany HA molecules (i.e., common determinants).

While reducing the present invention to practice, the present inventorshave uncovered that oligonucleotides (e.g., aptamers) designed to bindconserved sequences in the HA polypeptide can be utilized to preventvirus binding to host cells. As is illustrated hereinunder and in theexamples section, which follows, the present inventors, throughlaborious experimentation, have provided, for the first time, aptamernucleic acid molecules, which can be used to diagnose and treatinfluenza virus infection. Such aptamer molecules exhibit viralcross-reactivity and as such can be used as universal vaccines againstthe influenza virus.

Aptamers are nucleic acid sequences of tertiary structures, which areselected to specifically bind a polypeptide of interest and inhibit aspecified function thereof. Further description of aptamers andmechanism of action thereof is provided by Osborne, et al., Curr. Opin.Chem. Biol. 1997, 1(1): 5-9; and Patel, D. J., Curr. Opin. Chem. Biol.June 1997;1(1):32-46).

Thus, according to one aspect of the present invention there is provideda nucleic acid molecule including a polynucleotide sequence which iscapable of specifically binding a polypeptide participating in influenzavirus infection of cells.

The ability of the nucleic acid molecules of this aspect of the presentinvention to specifically bind a polypeptide which participates ininfluenza virus infection of cells allows the use thereof in influenzavirus infection therapy and diagnostics.

As used herein “a polypeptide which participates in influenza virusinfection of cells” refers to a polypeptide which is encoded by anorthomyxoviridea virus including type A-C influenza virus strains, ahost cell polypeptide or a peptide fragment thereof.

Examples of influenza virus polypeptides which participate in virusinfection of cells include but are not limited to hemagglutinin,neuraminidase, RNA-directed RNA polymerase core proteins including PB1,PB2 and PA, M1 and M2 matrix proteins, and NS proteins.

Examples of host cell polypeptides which participate in influenza virusinfection include but are not limited to mucoproteins containingterminal N-acetyl neuraminic acid (NANA=sialic acid) groups, HLAproteins and endocytic proteins sialic acid containing glycans andmucosal glycoproteins.

It will be appreciated that polypeptide targets of this aspect of thepresent invention are preferably viral, to maximize specificity of thenucleic acid molecules of the present invention and reduce cytotoxicitythereof. Accordingly, preferred polypeptide target sequences includeconserved amino acid sequences, which are shared by type A-C influenzaviruses. Nucleic acid molecules generated to bind such sequences can beused as universal vaccines.

Examples of conserved viral peptide targets are provided in Table 1,below.

TABLE 1 Viral peptide targets Influenza virus protein (amino acid SEQ IDcoordinates) Peptide sequence NO: Reference HA Ser-Lys-Ala-Phe-Ser-Asn-2 U.S. Pat. No. 4,474,757 (91-108) Cys-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu HA Pro-lys-tyr-val-lys-gln- 3 Rothbard (1998)Cell (306-318) asn-thr-leu-lys-leu-ala- 52(4):515-23 thr HACys-Pro-Lys-Tyr-Val-Lys- 4 Rothbard (1998) Cell (305-323)Gln-Asn-Thr-Leu-Lys-Leu 52(4):515-23 Ala-Thr-Gly-Met-Arg-Asn- Val NPSer-Ala-Ala-Phe-Glu-Asp- 5 Dyer and Middleton (335-350)Leu-Arg-Val-Leu-Ser-Phe- Histocompatability testing, a Ile-Arg-Gly-Tyrpractical approach Ed. Rickwood and Hames IRL Press Oxford (1993);Gulukota (1996) Biomolecular Engineering 13:81. NPGlu-Leu-Arg-Ser-Arg-Tyr- 6 Dyer and Middleton (380-393)Trp-Ala-Ile-Arg-Thr-Arg- Histocompatability testing, a Ser-Gly practicalapproach Ed. Rickwood and Hames IRL Press Oxford (1993); Gulukota (1996)Biomolecular Engineering 13:81. M1 Gly-Thr-His-Pro-Ser-Ser- 7 U.S. Pat.No. 5243030 (220-236) Ser-Ala-Gly-Leu-Lys-Asn- Asp-Leu-Leu-Glu-Asn M1Phe-Val-Gln-Asn-Ala-Leu-Asn-Gly- 8 U.S. Pat. No. 5243030 (79-104)Asn-Gly-Asp-Pro-Asn-Asn-Met-Asp- Arg-Ala-Val-Lys-Leu-Tyr-Arg-Lys-Leu-Lys M1 Phe-Thr-Leu-Thr-Val-Pro-Ser-Glu- 9 U.S. Pat. No. 5243030(64-80) Arg-Gly-Leu-Gln-Arg-Arg-Arg-Phe- Val M1Ala-Thr-Cys-Glu-Gln-Ile-ala-Asp- 10  U.S. Pat. No. 5243030 (149-169)Ser-Gln-His-Arg-Ser-His-Arg-Gln- Met-Val-ala-Thr-Thr

The nucleic acid molecules of this aspect of the present invention referto single stranded or double stranded DNA or RNA molecules or anymodifications thereof, which are capable of specifically binding thepolypeptide-targets described hereinabove. The nucleic acid molecules ofthis aspect of the present invention are interchangeably referred to as“aptamers”.

Typically, the nucleic acid molecules according to this aspect of thepresent are of varying length, such as 10-100 bases. It will beappreciated, though, that short nucleic acid molecules (e.g., 10-35bases) are preferably used for economical, manufacturing and therapeuticconsiderations, such as bioavailability (i.e., resistance to degradationand increased cellular uptake).

According to presently known embodiments of this aspect of the presentinvention, the nucleic acid molecules are preferably those set forth inSEQ ID NOs. 11 and 12 (i.e., A21 and A22).

As mentioned hereinabove, the nucleic acid molecules of this aspect ofthe present invention are preferably modified to obtain enhancedbioavailability and improved efficacy to the target polypeptide.Modifications include but are not limited to chemical groups whichincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction and fluxionality to the nucleic acid bases orto the entire molecule. Added or modified chemical groups are selectedto include conformationally flexible linkages, which conform to thetopology of the polypeptide target. Additionally, measures are takenthat the chemistry for the modification of the nucleic acid molecules ofthis aspect of the present invention allows for either trisphosphate(NTP) or phosphoramidite synthesis.

Thus, for example, nucleic acid molecules of this aspect of the presentinvention preferably include modifications which allow specificcross-linking to the target polypeptide to thereby form high affinitycompounds.

Appended cross-linking groups can contain hydrophobic, hydrophilic orcharged functionality. Cross-linking may be accomplished by theformation of imine, acetal, ester and disulfide linkages as well as byconjugate addition to α,β-unsaturated carbonyl linkers. Examples of2′-deoxyuridine nucleosides which are suitable for phosphoramiditesynthesis are shown in FIG. 1 a including small hydrophobic functionalgroups such as vinyl (group 1, FIG. 1 a), large hydrophobic functionalgroups such as pyrenyl (groups 13-14, FIG. 1 a) and carbonyl compoundswith varying degrees of side chain hydrophobicity (groups 3, 6-11, FIG.1 a).

Pyrimidine base modifications, such as RNA uridine nucleosidemodifications at position 5, can include hydrophobic groups which can beconjugated in the form of ketones [groups 17, 18 FIG. 1 a, Crouch (1994)Nucleosides Nucleotides 13:939-944], amides [groups, 24, 27, FIG. 1 a,Dewey (1995) J. Am. Chem. Soc. 117:8474-8475] and the like, which can beattached to either DNA or RNA nucleic acid molecules. It will beappreciated that amides can impart hydrogen bonding capabilities to theaptamer. In any case, as described hereinabove, cross-linking carbonylgroups can be attached to the 5-position of uridine (groups 15-18, FIG.1 a). It will be appreciated, though, that the expected reactivity ofcarbonyl linkers can differ significantly depending on the interface ofthe target polypeptide.

Examples of purine modifications are shown in FIG. 1 b. For examplehydrophobic substituents can be attached at the 8-position of RNA or DNApurine nucleosides (groups 28-30, FIG. 1 b). The degree of sterichindrance can be varied via amide linkages (groups 31, 33, 34, 37 and38, FIG. 1 b). Hydrophylic (group 35, FIG. 1 b) and charged (groups 36and 39, FIG. 1 b) groups may be appended to the 8 position of purinenucleosides. It will be appreciated that functional groups with knownaffinity to the target polypeptide can be attached to the 8 position ofthe purine base, such as a biotinylated nucleoside (group 40, FIG. 1 b).

Additional sites for modifications include but are not limited to the2′-position of RNA and the phosphodiester oxygens of RNA and DNA. A2′-position pyrimidine nucleoside modification can be effected accordingto Sebesta (1996) Tetrahedron 52:14385-14402; McGee (1996) TetrahedronLett. 37:1995-1998; McGee (1996) J. Org. Chem. 61:781-785. Essentially,amine linkers, such as hydroxyl amine linkers can be used to attachhydrophobic groups with different topologies (groups 41-43, 46 and 49,FIG. 1 c), hydrophilic groups (45 and 47, FIG. 1 c) and groupsexhibiting specific affinity to the target polypeptide (group 45, FIG. 1c).

As mentioned hereinabove, the nucleic acid molecules of this aspect ofthe present invention can also be modified to increase bioavailabilitythereof. The following illustrates non-limiting examples for suchmodifications.

The nucleic acid molecules of this aspect of the present invention maycomprise heterocylic nucleosides consisting of purines and thepyrimidines bases, bonded in a 3′ to 5′ phosphodiester linkage.

Preferably used nucleic acid molecules are those modified in eitherbackbone, internucleoside linkages or bases, as is broadly describedhereinunder. Such modifications can oftentimes facilitateoligonucleotide uptake and resistance to intracellular conditions.

Array of synthetic chemistry is available for modification ofnucleosides which may be converted to either NTPs or phosphoramiditereagents. For further details see Eaton and Pieken (1995) Annu. Rev.Biochem. 64:837-863.

Specific examples of nucleic acid molecules useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. NOS:,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050.

Preferred modified nucleic acid backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid form also be used.

Alternatively, modified nucleic acid backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkylor cycloalkyl intemucleoside linkages, or one or more short chainheteroatomic or heterocyclic intemucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other nucleic acid molecules which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such an nucleic acidsequence mimetic, includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No: 6,303,374.

Nucleic acid molecules of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No: 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Such bases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2 C. [Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278] and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the nucleic acid molecules of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates, which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, asdisclosed in U.S. Pat. No: 6,303,374.

It is not necessary for all positions in a given oligonucleotidemolecule to be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single compound oreven at a single nucleoside within an oligonucleotide.

As is illustrated in the Examples section, which follows, the presentinventors have conclusively shown that the nucleic acid molecules of thepresent invention are capable of preventing influenza virus infection ofcells in vitro and in vivo. Furthermore, the ability of the nucleic acidmolecules of the present invention to inhibit viral spread followingviral challenging, suggests the use of the nucleic acid molecules of thepresent invention in anti-influenza prophylactic and therapeuticapplications.

Thus, according to another aspect of the present invention there isprovided a method of treating influenza virus infection.

As used herein the term “treating” refers to preventing influenza virusinfection or substantially reducing (i.e., alleviating or diminishing)symptoms associated with influenza virus infection.

The method is effected by providing to a subject in need thereof, atherapeutically effective amount of the nucleic acid molecule of thepresent invention described hereinabove.

As used herein “a subject in need thereof” refers to a subject sufferingfrom influenza-virus associated symptoms or at risk of contractinginfluenza. Examples of such subjects include but are not limited topeople aged 65 or over; people with chronic diseases of the heart, lungor kidneys, diabetes, immuno-suppression, or severe forms of anemiaresidents of nursing homes and other chronic-care facilities, childrenand teenagers taking aspirin therapy and who may therefore be at riskfor developing reye syndrom reye syndrome after an influenza infection,and those in close or frequent contact with anyone at high risk.

Preferably, the nucleic acid molecules of the present invention areprovided at a concentration of between, 0.1-150 μg/Kg body weight,preferably 1-100 μg/Kg body weight, more preferably 1-50 μg/Kg bodyweight and even more preferably 1-15 μg/Kg body weight.

The nucleic acid molecule (i.e., active ingredient) of the presentinvention can be provided to the subject per se, or as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism. Herein the term “activeingredient” refers to the preparation accountable for the biologicaleffect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Since activity of aptamers is directly correlated with a molecularweight thereof, measures are taken to conjugate the nucleic acidmolecules of the present invention to high molecular weight carriers.Such high molecular weight carriers include, but are not limited to,polyalkylene glycol and polyethylene glycol (PEG), which arebiocompatible polymers with a wide range of solubility in both organicand aqueous media (Mutter et al. (1979).

Alternatively, microparticles such as microcapsules or cationic lipidscan serve as the pharmaceutically acceptable carriers of this aspect ofthe present invention.

As used herein, microparticles include liposomes, virosomes,microspheres and microcapsules formed of synthetic and/or naturalpolymers. Methods for making microcapsules and microspheres are known tothe skilled in the art and include solvent evaporation, solvent casting,spray drying and solvent extension. Examples of useful polymers whichcan be incorporated into various microparticles include polysaccharides,polyanhydrides, polyorthoesters, polyhydroxides and proteins andpeptides.

Liposomes can be generated by methods well known in the art such asthose reported by Kim et al., Biochim. Biophys. Acta, 728:339-348(1983); Liu et al., Biochim. Biophys. Acta, 1104:95-101 (1992); and Leeet al., Biochim. Biophys. Acta, 1103:185-197 (1992); Wang et al.,Biochem., 28:9508-9514 (1989). Alternatively, the nucleic acid moleculesof this aspect of the present invention can be incorporated withinmicroparticles, or bound to the outside of the microparticles, eitherionically or covalently.

Cationic liposomes or microcapsules are microparticles that areparticularly useful for delivering negatively charged compounds such asthe nucleic acid molecules of this aspect of the present invention,which can bind ionically to the positively charged outer surface ofthese liposomes. Various cationic liposomes are known to be veryeffective at delivering nucleic acids or nucleic acid-protein complexesto cells both in vitro and in vivo, as reported by Felgner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7417 (1987); Felgner, Advanced DrugDelivery Reviews, 5:163-187 (1990); Clarenc et al., Anti-Cancer DrugDesign, 8:81-94 (1993). Cationic liposomes or microcapsules can begenerated using mixtures including one or more lipids containing acationic side group in a sufficient quantity such that the liposomes ormicrocapsules formed from the mixture possess a net positive chargewhich will ionically bind negatively charged compounds. Examples ofpositively charged lipids which may be used to produce cationicliposomes include the aminolipid dioleoyl phosphatidyl ethanolamine(PE), which possesses a positively charged primary amino head group;phosphatidylcholine (PC), which possess positively charged head groupsthat are not primary amines; andN[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (“DOTMA,” see Felgneret al., Proc. Natl. Acad. Sci USA, 84:7413-7417 (1987); Felgner et al.,Nature, 337:387-388 (1989); Felgner, Advanced Drug Delivery Reviews,5:163-187 (1990)).

As mentioned hereinabove the pharmaceutical compositions of this aspectof the present invention may further include excipients. The term“excipient”, refers to an inert substance added to a pharmaceuticalcomposition to further facilitate administration of an activeingredient. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Pharmaceutical compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert.

It will be appreciated that the nucleic acid molecules of the presentinvention can also be expressed from a nucleic acid constructadministered to the individual subject employing any suitable mode ofadministration, described hereinabove. Alternatively, the nucleic acidconstruct is introduced into a suitable cell via an appropriate deliveryvehicle/method (transfection, transduction, and the like) and anexpression system as needed and then the modified cells are expanded inculture and returned to the individual.

To enable cellular expression of RNA nucleic acid molecules of thepresent invention, the nucleic acid construct of the present inventionfurther includes at least one cis acting regulatory element. As usedherein, the phrase “cis acting regulatory element” refers to apolynucleotide sequence, preferably a promoter, which binds a transacting regulator and regulates the transcription of a coding sequencelocated downstream thereto.

Any available promoter can be used by the present methodology. Preferredpromoters for use in aptamer expression vectors include the pol IIIpromoters such as the human small nuclear U6 gene promoter and tRNA genepromoters. The use of U6 gene transcription signals to produce short RNAmolecules in vivo is described by Noonberg et al., Nucleic Acids Res.22:2830-2836 (1994), and the use of tRNA transcription signals isdescribed by Thompson et al., Nucleic Acids Res., 23:2259-2268 (1995).

It will be appreciated that many pol III promoters are internal and arelocated within the transcription unit such that pol III transcriptsinclude promoter sequences. To be useful for expression of aptamermolecules, these promoter sequences should not interfere with thestructure or function of the aptamer. Therefore a preferred RNA pol IIIRNA promoter is the U6 gene promoter which is not internal [Kunkel andPederson, Nucleic Acids Res, 17:7371-7379 (1989); Kunkel et al., Proc.Natl. Acad Sci. USA 83:8575-8579 (1986); Reddy et al., J. Biol. Chem.262:75-81 (1987)]. Suitable pol III promoter systems useful forexpression of aptamer molecules are described by Hall et al., Cell29:3-5 (1982), Nielsen et al., Nucleic Acids Res. 21:3631-3636 (1993),Fowlkes and Shenk, Cell 22:405-413 (1980), Gupta and Reddy, NucleicAcids Res. 19:2073-2075 (1991), Kickhoefer et al., J. Biol. Chem.268:7868-7873 (1993), and Romero and Blackburn, Cell 67:343-353 (1991).The use of pol III promoters for expression of RNA molecules is alsodescribed in WO 95/23225 by Ribozyme Pharmaceuticals, Inc.

Other promoters useful for expressing the aptamers of the presentinvention include, for example, the genomes of viruses such as: polyoma,Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus andmost preferably cytomegalovirus, or from heterologous mammalianpromoters, e.g. beta actin promoter. The early and late promoters of theSV40 virus can be obtained as an SV40 restriction fragment which alsocontains the SV40 viral origin of replication [Fiers et al., Nature,273: 113 (1978)]. The immediate early promoter of the humancytomegalovirus can be obtained as a HindIII E restriction fragment(Greenway, P. J. et al., Gene 18: 355-360 (1982)). It will beappreciated that promoters from the host cell or related species alsocan also be used.

The constructs of the present methodology preferably further include anappropriate selectable marker and/or an origin of replication.Preferably, the construct utilized is a shuttle vector, which canpropagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation in cells, or integration in a tissue ofchoice. The construct according to the present invention can be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer aredescribed by Lasic D., Liposomes: From Physics to Applications,Elsevier: Amsterdam, 1993.

Preferably, cationic lipids are used in combination with a neutral lipidin equimolar amounts as described hereinabove. Neutral lipids of use intransfection complexes include, for example, dioleoylphosphatidylethanolamine (DOPE), Hui et al., Biophys. J. (71)590-599(1996); cholesterol, Liu et al., Nat. Biotech. 15:167-173 (1997).

Typically a lipid mixtures is prepared in chloroform, dried, andrehydrated in, e.g., 5% dextrose in water or a physiologic buffer toform liposomes. The resulting liposomes are mixed with a nucleic acidsolution with constant agitation to form the cationic lipid-nucleic acidtransfection complexes. Preferred transfection complex size forintravenous administration is from 50 to 5000 nm, most preferably from100 to 400 nm.

It will be appreciated that DNA/lipid complexes are preferably preparedat a DNA concentration of about 0.625 mg/ml. The dose delivered is fromabout 10 .mu.g to about 2 mg per gram of body weight. Repeat doses maybe delivered at intervals of from about 2 days to about 2 months.

The most preferred constructs for in-vivo use according to presentlyknown embodiments are viruses, most preferably adenoviruses, AAV,lentiviruses, or retroviruses. A viral construct such as a retroviralconstruct includes at least one transcriptional promoter/enhancer orlocus-defining element(s), or other elements that control geneexpression by other means such as alternate splicing, nuclear RNAexport, or post-translational modification of messenger. Such vectorconstructs also include a packaging signal, long terminal repeats (LTRs)or portions thereof, and positive and negative strand primer bindingsites appropriate to the virus used, unless it is already present in theviral construct. In addition, such a construct typically includes asignal sequence for secretion of the peptide or antibody from a hostcell in which it is placed. Preferably the signal sequence for thispurpose is a mammalian signal sequence. Optionally, the construct mayalso include a signal that directs polyadenylation, as well as one ormore restriction sites and a translation termination sequence. By way ofexample, such constructs will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof.

Preferred modes for in-vivo nucleic acid delivery protocols are providedin Somia and Verma (2000) Nature Reviews 1:91-99, Isner (2002)Myocardial gene therapy Nature 415:234-239; High (2001) Gene therapy: a2001 perspective. Haemophilia 7:23-27; and Hammond and McKirnan (2001)Angiogenic gene therapy for heart disease: a review of animal studiesand clinical trials. 49:561-567.

Prior to, concomitant with or following providing the nucleic acidmolecule of the present invention an agent can be provided to thesubject.

An agent can be a molecule which facilitates prevention or treatment ofinfluenza infection or clinical conditions associated with influenzainfection such as pneumonia. Examples of agents, according to thisaspect of the present invention include, but are not limited to,immunomodulatory agents (e.g., antibodies), antibiotics, antiviral agent(e.g., amantidine), antisense molecules, rybosymes and the like.

The antibody-like nature (i.e., specific binding to a polypeptidetarget) of the nucleic acid molecules of the present invention, allowsthe agents described hereinabove to be specifically targeted to aninfectious tissue upon attachment to the administered nucleic acidmolecule or to a lipid carrier containing same.

For example an antisense molecule directed at an anfluenza viruspolypeptide (further described in the background section) can betargeted using the apatmeric sequences of the present invention.“Chimeric” antisense molecules”, are oligonucleotides, which contain twoor more chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetpolynucleotide. An additional region of the oligonucleotide may serve asa substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.An example for such include RNase H, which is a cellular endonucleasewhich cleaves the RNA strand of an RNA:DNA duplex. Activation of RNaseH, therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense molecules of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, as described above. Representative U.S. patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922, each of which is herein fully incorporated byreference.

Alternatively a ribozyme sequence can be targeted using the nucleic acidmolecules of the present invention. Ribozymes are being increasinglyused for the sequence-specific inhibition of gene expression by thecleavage of mRNAs. Several ribozyme sequences can be fused to theoligonucleotides of the present invention. These sequences include butare not limited ANGIOZYME specifically inhibiting formation of theVEGF-R (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway, and HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, (RibozymePharmaceuticals, Incorporated—WEB home page).

Optionally, “DNAzymes” can be targeted using the methodology of thepresent invention [Breaker, R. R. and Joyce, G. Chemistry and Biology(1995);2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA1997;943:4262]. DNAzymes are single-stranded, and cleave both RNA. Ageneral model (the “10-23” model) for the DNAzyme has been proposed.“10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions (Santoro, S. W. &Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes seeKhachigian, L M Curr Opin Mol Ther 2002;4:119-21).

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al ,20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). Inanother application, DNAzymes complementary to bcr-abl oncogenes weresuccessful in inhibiting the oncogenes expression in leukemia cells, andlessening relapse rates in autologous bone marrow transplant in cases ofCML and ALL.

Methods of nucleic acid-lipid coupling are well known in the art anddescribed in U.S. Pat. No. 5,756,291.

For example, Asseline, U. et al. [Proc Natl Acad Sci 81, 3297-3301(1984)] describes the covalent linking of an intercalating agent via apolymethylene linker through a 3′-phosphate group. Mori, K. et al. (FEBSLetters 249:213-218 (1989)) describes the covalent attachment of groupsvia a methylene linker at the 5′-terminus of oligonucleotides. PCTapplication WO89/05853 published Jun. 29, 1989, the entire disclosure ofwhich is hereby incorporated by reference, describes a variety ofmethods for formation of conjugates between nucleotide sequences andchelating agents; the chelating agent is joined to the nucleotidessequence by either a covalent bond or a linking unit derived from apolyvalent functional group.

Thus, the aptamers or modified aptamers of the invention may be usedalone in therapeutic applications or may be used for targeting agents todeliver pharmaceuticals or toxins to desired targets.

The ability of the nucleic acid molecules of the present invention tospecifically bind polypeptides of the influenza virus allows the usethereof in diagnostic applications.

To date, a number of tests are available for the diagnosis of influenzaA and B. A traditional approach for identifying influenza viruses inbiological samples involves cell culturing, thereby providing highlysensitive and specific detection of viral infection. However, thisapproach is significantly limited by the time required for cellculturing and identification of influenza virus can range between 2 and10 days, thus making it ineffective in guiding the physician to anappropriate therapy. Since influenza virus infection is normallyself-limited, diagnosis must be rapid if therapy is to be effective.Thus, cell culture methods are used only for providing retrospectiveepidemiological information.

Other influenza diagnostic methods include the use of monoclonalimmunofluorescence assays [Spada, B. et al., J. Virol. Methods, (1991)33: 305] and enzyme-linked immunoassay [EIA, Ryan-Poirier, K. A. et al.,J. Clin. Microbiol., (1992) 30: 1072]. However, not only are thesemethods limited to the identification of type A influenza virusinfection, but they require considerable technical expertise, and resultin high levels of false-positive.

Thus, according to yet another aspect of the present invention there isprovided a method of identifying influenza virus in a biological sample.

As used herein a biological sample refers to any body sample such asblood, l s spinal fluid, pleural fluid, respiratory fluids and nasalaspirates. Methods of obtaining body fluids from vertebrates are wellknown in the art. For example, a nasal wash can be obtained as describedin Henrickson, J. Viol. Methods, 46:189-206, 1994 or Hall and Douglas,J: Infect. Dis., 131:1-5, 1975.

The method is effected by contacting the biological sample with anucleic acid molecule including a polynucleotide sequence capable ofspecifically binding an influenza virus polypeptide, describedhereinabove.

The nucleic acid molecules of the present invention can be attached to asolid substrate, such as described hereinbelow.

Contacting is effected under conditions which allow the formation of apolyepeptide-nucleic acid molecule duplex.

Duplexes are preferably washed to remove any non-specifically boundpolypeptides allowing only those nucleic acid molecules specificallybound within the complexes to be detected.

Polypeptide-bound nucleic acid molecules in the biological sample aredetected to thereby identify the influenza infection.

In general monitoring of polypeptide-nucleic acid molecule complexes iswell known in the art and may be effected as described hereinabove.These approaches are generally based on the detection of a label ormarker, such as described hereinbelow.

Preferably, detection of an infected sample is effected by comparison toa normal sample, which is not infected with an influenza virus.

To generate the nucleic acid molecules of the present invention, arobust selection approach is preferably employed.

Thus, according to an additional aspect of the present invention thereis provided a method of generating a nucleic acid molecule, which iscapable of inhibiting influenza virus infection of cells.

The method is effected as follows.

First, a plurality of nucleic acid molecules are contacted with apolypepitde target, which participates in influenza virus infection ofcells as described hereinabove.

Following duplex formation (i.e., a non-Watson Crick complementationbetween the polypeptide target and the nucleic acid molecules), at leastone nucleic acid molecule of the plurality of nucleic acid moleculeswhich is capable of specifically binding the polypeptide is identified.

Finally, polypeptide bound nucleic acid molecules are isolated tothereby generate the molecule which is capable of inhibiting influenzavirus infection.

Double stranded DNA molecules can be generated from a library ofoligonucleotide sequences including a randomized polynucleotide sequenceflanked by two defined nucleotide sequences which can be used forpolymerase chain reaction (PCR) primer binding. The library is amplifiedto yield double-stranded PCR products [Bielinska (1990) Science250(4983):997-1000]. The randomized sequences can be completelyrandomized (i.e., the probability of finding a base at any positionbeing 1:4) or partially randomized (i.e., the probability of finding abase at any position is selected at any level between 0-100%).

For preparation of single stranded aptamers, the down stream primer isbiotinylated at the 5′ end and PCR products are applied to an avidinagarose column. Single stranded DNA sequences are recovered by elutionwith e weakly basic buffer.

Single stranded RNA molecules can be generated from an oligonucleotidesequence library, which is amplified to yield double-stranded PCRproducts containing a T7 bacteriophage polymerase promoter site. RNAmolecules can then be produced by in vitro transcription using T7 RNApolymerase.

The nucleic acid molecules of this aspect of the present invention canbe generated from naturally-occurring nucleic acids or fragmentsthereof, chemically synthesized nucleic acids, enzymatically synthesizednucleic acids or nucleic acid molecules made by a combination of theforegoing techniques

The library of this aspect of the present invention is generatedsufficiently large to provide structural and chemical coverage ofselected nucleic acid modifications described hereinabove.

Typically, a randomized nucleic acid sequence library according to thisaspect of the present invention includes at least 10¹⁴ sequencevariants.

Nucleic acid modifications can be effected prior to incubation with thetarget polypeptide. In this case, although screening is effected on thefinal modified aptamer, modification is restricted not to interfere withany process, such as an enzymatic process (e.g., transcription), whichtakes place during the screening.

Alternatively, a nucleic acid molecule can be modified followingselection (i.e., isolation of a polypeptide bound nucleic acidmolecule). Thus, a wide range of functional groups can be usedsimultaneously. In this case, electrospray ionization mass spectrometry(ESI-MS) can be used to elucidate the right functional group [Pomerantz(1996) Anal. Chem. 68:1989-1999].

In any case, once nucleic acid molecules are obtained they are contactedwith the polypeptide target, as mentioned hereinabove.

Incubation of the nucleic acid molecules with the target polypeptide ofthis aspect of the present invention is preferably effected underphysiological conditions. As used herein the phrase “physiologicalconditions” refers to salt concentration and ionic strength in anaqueous solution, which characterize fluids found in the metabolism ofvertebrate animal subjects which can be infected with influenza virus,also referred to as physiological buffer or physiological saline. Forexample physiological fluids of human subjects are represented by anintracellular pH of 7.1 and salt concentrations (in mM) of sodium 3-15;potassium 140; magnesium 6.3; Calcium 10⁻⁴; Chloride 3-15, and anextracellular pH of 7.4 and salt concentrations (in mM) of sodium 145;potassium 3; Magnesium 1-2; Calcium 1-2; and Chloride 110.

The nucleic acid molecules can be incubated with the target polypeptideeither in solution or when bound to a solid substrate.

It will be appreciated that some of the above-described basemodifications can be used as intermediates for attaching the nucleicacid molecule to a solid substrate. For example, the modified uridineshown in group 48 of FIG. 1 c, can serve as a common intermediate whichmay be further modified by substitution of the imidazole with a widevariety of hydrophobic, hydrophilic, charged and cross linking groups,prior to activation as the phosphoramidite reagent used in solid phasesynthesis

Methods for attaching nucleic acid molecules to solid substrates areknown in the art including but not limited to glass-printing, describedgenerally by Schena et al., 1995, Science 270:467-47, photolithographictechniques [Fodor et al. (1991) Science 251:767-773], inkjet printing,masking and the like.

Typically, a control sample is included to select against nucleic acidmolecules which bind to non-target substances such as the solid supportand/or non target epitopes.

Separation of unbound nucleic acid sequences and identification of boundnucleic acid sequences can be effected using methods well known in theart. Examples include, but are not limited to, selective elution,filtration, electrophoresis and the like (see U.S. Pat. No. 5,756,291).

Alternatively, bound aptameric molecules can be identified by imaging.For example, optical microscopy using bright field, epi-fluorescence orconfocal methods, or scanning probe microscopy can be used to identify apolypeptide bound nucleic acid molecule (see U.S. Pat. No. 6,287,765).To facilitate visualization, nucleic acid molecules or polypeptides arepreferably labeled using any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art.

The following illustrates a number of labeling methods suitable for usein the present invention. For example, nucleic acid molecules of thepresent invention can be labeled subsequent to synthesis, byincorporating biotinylated dNTPs or rNTP, or some similar means (e.g.,photo-cross-linking a psoralen derivative of biotin to RNAs), followedby addition of labeled streptavidin (e.g., phycoerythrin-conjugatedstreptavidin) or the equivalent. Alternatively, fluorescent moieties areused, including but not limited to fluorescein, lissamine,phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992),Academic Press San Diego, Calif.]. Alternatively, a radioactive label isused [Zhao et al. (1995) Gene 156:207]. However, because of scatteringof radioactive particles, and the consequent requirement for widelyspaced binding sites, the use of fluorophores rather than radioisotopesis more preferred.

It will be appreciated that the intensity of signal produced in any ofthe detection methods described hereinabove may be analyzed manually orusing a software application and hardware suited for such purposes.

Isolation of an aptamer sequence (i.e., polypeptide-bound nucleic acid)typically involves sequence amplification such as by PCR. Amplificationmay be conducted prior to, concomitant with or following separation fromthe target polypeptide. The PCR method is well known in the art anddescribed in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, and4,800,159 as well as Methods in Enzymology (1987) 155:335-350. It willbe appreciated that if RNA molecules are used, the amplified DNAsequences are transcribed into RNA.

Other methods of amplification may be employed including standardcloning, ligase chain reaction and the like (see U.S. Pat. No.4,957,858). For example, once an aptamer is identified, linkers may beattached to each side to facilitate cloning into standard vectors.Single stranded or double stranded aptamers, may be cloned andrecovered.

The recovered nucleic acid molecule, in the original single-stranded orduplex form, can then be used for iterative rounds of selection andamplification (i.e., target polypeptide binding). Typically, followingthree to six rounds of selection/amplification, nucleic acid moleculeswhich bind with a preferred affinity of nM to M range can be obtained.

It will be appreciated that methods for identifying nucleic acidmolecules capable of specifically binding polypeptide targets are knownin the art [e.g., U.S. Pat. No. 5,270,163, Ellington and Szostak (1990)Nature 346:818-822, Bock et al. (1992) Nature 255:564-566, Wang et al.(1993) Biochemistry 32:1899-1904, and Bielinska et al. (1990) Science250:997-1000]. For example, U.S. Pat. No. 5,270,163 discloses a methodreferred to as SELEX (Systematic Evolution of Ligands by ExponentialEnrichment) for the identification of nucleic acid ligands as follows. Acandidate mixture of single-stranded nucleic acids having regions ofrandomized sequence is contacted with a target compound and thosenucleic acids having an increased affinity to the target are partitionedfrom the remainder of the candidate mixture. The partitioned nucleicacids are amplified to yield a ligand enriched mixture. Bock andco-workers describe a method for identifying oligomer sequences thatspecifically bind target biomolecules involving complexation of thesupport-bound target molecule with a mixture of oligonucleotidescontaining random sequences and sequences that can serve as primers forPCR [Bock et al. (1992) Nature 255:564-566]. The target-oligonucleotidecomplexes are then separated from the support and the uncomplexedoligonucleotides, and the complexed oligonucleotides are recovered andsubsequently amplified using PCR. The recovered oligonucleotides may besequenced and subjected to successive rounds of selection usingcomplexation, separation, amplification and recovery.

Alternatively, the nucleic acid sequences of the present invention canbe generated by rational drug design.

Rational drug design is a potent means of identifying enzyme inhibitorswhich, for example, has notably been used to identify HIV protease (Lamet al., 1994. Science 263, 380; Wlodawer et al., 1993. Ann Rev Biochem.62, 543; Appelt, 1993. Perspectives in Drug Discovery and Design 1, 23;Erickson, 1993. Perspectives in Drug Discovery and Design 1, 109), andbcr-abl tyrosine kinase inhibitors (Mauro M J. et al., 2002. J ClinOncol. 20, 325-34) used to provide the first effective pharrnacologicalcures for human acquired immunodeficiency syndrome (AIDS) caused byhuman immunodeficiency virus (HIV)), and a human cancer (chronic myeloidleukemia), respectively.

To identify a putative aptamer sequence via rational drug design byscreening a nucleic acid sequence structure database (“3D database”),software employing “scanner” type algorithms employ atomic coordinatesdefining the three-dimensional structure of a binding pocket of amolecule, such as the sialic acid receptor binding pocket of theinfuenza hemagglutinin polypeptide (amino acid coordinates 116-261 ofGenBank Accession No. AF092062), and of a nucleic acid sequencestructure stored in the database to computationally model the “docking”of the screened aptamer structure with the binding pocket so as toqualify the binding of the binding pocket with the aptamer structure.Iterating this process with each of a plurality of putative aptamerstructures stored in the database therefore enables computationalscreening of such a plurality to identify a chemical structurepotentially having a desired binding interaction with the bindingpocket, and hence the putative inhibitor.

Examples of nucleic acid structure databases for identifying the nucleicacid molecule of the present invention include the RNA structuredatabase (www.RNABase.org) and the NDB database (http://www.imb-jena.de/RNA.html#Databases).

Alternatively, a refined aptamer sequence can be elucidated by modifyinga known aptamer structure (e.g., A22, SEQ ID NO: 12) using a softwarecomprising “builder” type algorithms which utilizes a set of atomiccoordinates defining a three-dimensional structure of the binding pocketand the three-dimensional structures of the basic aptamer (e.g., A22) tocomputationally assemble a refined aptamer. Ample guidance forperforming rational drug design via software employing such “scanner”and “builder” type algorithms is available in the literature of the art(for example, refer to: Halperin I. et al., 2002. Proteins 47, 409-43;Gohlke H. and Klebe G., 2001. Curr Opin Struct Biol. 11, 231-5; Zeng J.,2000. Comb Chem High Throughput Screen. 3, 355-62; and RACHEL: Theory ofdrug design, http://www.newdrugdesign.com/Rachel_Theory.htm#Software),and described in further detail hereinbelow.

Criteria employed by software programs used in rational drug design toqualify the binding of screened aptamer structures with binding pocketsinclude gap space, hydrogen bonding, electrostatic interactions, van derWaals forces, hydrophilicity/hydrophobicity, etc. Generally, the greaterthe contact area between the screened molecule and the polypeptidebinding region, the lower the steric hindrance, the lower the “gapspace”, the greater the number of hydrogen bonds, and the greater thesum total of the van der Waals forces between the screened molecule andthe polypeptide binding region of, the greater will be the capacity ofthe screened molecule to bind with the target polypeptide. The “gapspace” refers to unoccupied space between the van der Waals surface of ascreened molecule positioned within a binding pocket and the surface ofthe binding pocket defined by amino acid residues in the binding pocket.Gap space may be identified, for example, using an algorithm based on aseries of cubic grids surrounding the docked molecule, with auser-defined grid spacing, and represents volume that couldadvantageously be occupied by a modifying the docked a[tamer positionedwithin the binding region of the polypeptide target.

Contact area between compounds may be directly calculated from thecoordinates of the compounds in docked conformation using the MS program(Connolly M L., 1983. Science 221, 709-713).

Suitable software employing “scanner” type algorithms include, forexample, docking software such as GRAM, DOCK, or AUTODOCK (reviewed inDunbrack et al., 1997. Folding and Design 2, 27), AFFINITY software ofthe INSIGHTII package (Molecular Simulations Inc., 1996, San Diego,Calif.), GRID (Goodford P J., 1985. “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules”, J. Med. Chem. 28, 849-857; GRID is availablefrom Oxford University, Oxford, UK), and MCSS (Miranker A. and KarplusM., 1991. “Functionality Maps of Binding Sites: A Multiple CopySimultaneous Search Method”, Proteins: Structure Function and Genetics11, 29-34; MCSS is available from Molecular Simulations, Burlington,Mass.).

The AUTODOCK program (Goodsell D S. and Olson A J., 1990. Proteins:Struct Funct Genet. 8, 195-202; available from Scripps ResearchInstitute, La Jolla, Calif.) helps in docking screened molecules tobinding pockets in a flexible manner using a Monte Carlo simulatedannealing approach. The procedure enables a search without biasintroduced by the researcher. This bias can influence orientation andconformation of a screened molecule in the targeted binding pocket.

The DOCK program (Kuntz I D. et al., 1982. J Mol Biol. 161, 269-288;available from University of California, San Francisco), is based on adescription of the negative image of a space-filling representation ofthe binding pocket, and includes a force field for energy evaluation,limited conformational flexibility and consideration of hydrophobicityin the energy evaluation.

Modeling or docking may be followed by energy minimization with standardmolecular mechanics force fields or dynamics with programs such asCHARMM (Brooks B R. et al., 1983. J Comp Chem. 4, 187-217) or AMBER(Weiner S J. et al., 1984. J Am Chem Soc. 106, 765-784).

As used herein, “minimization of energy” means achieving an atomicgeometry of a chemical structure via systematic alteration such that anyfurther minor perturbation of the atomic geometry would cause the totalenergy of the system as measured by a molecular mechanics force-field toincrease. Minimization and molecular mechanics force fields are wellunderstood in computational chemistry (for example, refer to Burkert U.and Allinger N L., “Molecular Mechanics”, ACS Monograph 177, pp. 59-78,American Chemical Society, Washington, D.C. (1982)).

Programs employing “builder” type algorithms include LEGEND (NishibataY. and Itai A., 1991. Tetrahedron 47, 8985; available from MolecularSimulations, Burlington, Mass.), LEAPFROG (Tripos Associates, St. Louis,Mo.), CAVEAT (Bartlett, P A. et al., 1989. Special Pub Royal Chem Soc.78, 182-196; available from University of California, Berkeley), HOOK(Molecular Simulations, Burlington, Mass.), and LUDI (Bohm H J., 1992.J. Comp Aid Molec Design 6, 61-78; available from Biosym Technologies,San Diego, Calif.).

The CAVEAT program suggests binding molecules based on desired bondvectors. The HOOK program proposes docking sites by using multiplecopies of functional groups in simultaneous searches. LUDI is a programbased on fragments rather than on descriptors which proposes somewhatlarger fragments to match with a binding pocket and scores its hitsbased on geometric criteria taken from the Cambridge Structural Database(CSD), the Protein Data Bank (PDB) and on criteria based on bindingdata. LUDI may be advantageously employed to calculate the inhibitionconstant of a docked chemical structure. Inhibition constants (Kivalues) of compounds in the final docking positions can be evaluatedusing LUDI software.

During or following rational drug design, docking of an intermediatechemical structure or of a putative aptamer with the binding pocket maybe visualized via structural models, such as three-dimensional models,thereof displayed on a computer screen, so as to advantageously allowuser intervention during the rational drug design to optimize a chemicalstructure.

Software programs useful for displaying such three-dimensionalstructural models, include RIBBONS (Carson, M., 1997. Methods inEnzymology 277, 25), O (Jones, T A. et al., 1991. Acta Crystallogr. A47,110), DINO (DINO: Visualizing Structural Biology (2001)http://www.dino3d.org); and QUANTA, INSIGHT, SYBYL, MACROMODE, ICM,MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. ApplCrystallogr. 24, 946).

Other molecular modeling techniques may also be employed in accordancewith this invention (for example, refer to: Cohen N C. et al, 1990.“Molecular Modeling Software and Methods for Medicinal Chemistry”, J.Med. Chem. 33, :883-894; Navia M. A. and Murcko M. A., 1992. “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology 2, 202-210). For example, where the structures of test compoundsare known, a model of the test compound may be superimposed over themodel of the structure of the invention. Numerous methods and techniquesare known in the art for performing this step, any of which may be used(for example, refer to: Farmer P. S., “Drug Design”, Ariens E J. (ed.),Vol. 10, pp 119-143 (Academic Press, New York, 1980); U.S. Pat. No.5,331,573; U.S. Pat. No. 5,500,807; Verlinde C., 1994. Structure 2,577-587; and Kuntz I D., 1992. Science 257, 1078-108).

In any case once putative aptamer sequences are identified they areexamined for specific binding to the target polypeptide, which can beeffected using a number of biochemical methods known in the art, such asa band shift assay (U.S. Pat. No. 5,756,291) and affinity chromatography[Schott, H., Affinity Chromatography, (Marcel Dekker, Inc., New York,1984)].

Alternatively or additionally, the nucleic acid sequences of the presentinvention are tested for inhibiting influenza virus infection in vitrosuch as in MDCK cultured cell line, or in vivo as further described inExample 2 (in vitro) and Example 3 (in vivo) of the Examples sectionwhich follows.

As described hereinabove, an important constituent in aptamer design isselection of the polypeptide target. It is appreciated that peptidesused for selecting the aptamer molecules of the present invention can beused as potent tools in influenza related therapeutic and diagnosticapplications (13).

Thus, according to yet an additional aspect of the present inventionthere is provided a polypeptide useful for vaccination against aninfluenza virus (i.e., the orthomyoxiviruses).

The polypeptide of this aspect of the present invention includes anamino acid sequence which is preferably at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 92%, at least 94% ormore, say 95%-100%, homologous to SEQ ID NO: 13 as determined using theBestFit software of the Wisconsin sequence analysis package, utilizingthe Smith and Waterman algorithm, where gap creation penalty equals 8and gap extension penalty equals 2 reflecting the conservation of thepolypeptide among the various influenza strains and including functionalhomologues as well.

The polypeptide of this aspect of the present invention does not includethe HA2 domain of influenza virus.

Preferably, the polypeptide of the present invention includes an aminoacid sequence defined by amino acid coordinates 116-261 of SEQ ID NO: 14which encompass the globular region of the influenza HA which mediatesbinding to host cell determinants such as the sialic acid receptors.

More preferably the polypeptide of the present invention includes anamino acid sequence defined by amino acid coordinates 116-245 of SEQ IDNO: 15 which encompass a further minimal globular region of theinfluenza HA.

Since the receptor binding pocket of influenza HA polypeptide is mostlyunexposed to the immune system due to conformational restrictions, thepolypeptide of this aspect of the present invention, preferably furtherincludes additional antigenic epitopes such as defined by amino acidcoordinates 91-261 of SEQ ID NO: 1 [McEwen (1992) Vaccine 10:405-411;Muller (1982) Proc. Natl. Acad. Sci. USA 79:569-573; Shapira (1985) J.Immunopharmacol. 7:719-723].

It will be appreciated that other antigenic epitopes, which arepreferably conserved can be included in the polypeptide of the presentinvention, such as provided in Table 1, hereinabove.

Preferably, the polypeptide of this aspect of the present invention isas set forth in SEQ ID NOs: 13-15.

Alternatively, the polypeptide of this aspect of the present inventionincludes the amino sequence set forth in SEQ ID NOs: 13-15.

The term “polypeptide” as used herein encompasses native peptides(either degradation products, synthetically synthesized peptides orrecombinant peptides) and peptidomimetics (typically, syntheticallysynthesized peptides), as well as as peptoids and semipeptoids which arepeptide analogs, which may have, for example, modifications renderingthe peptides more stable while in a body or more capable of penetratinginto cells. Such modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH2—NH, CH2—S, CH2—S═O,O═C—NH, CH2—O, CH2—CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2—), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2—NH—), hydroxyethylene bonds (—CH(OH)—CH2—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2—CO═), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc.).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-arninoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 2 and 3 below list naturally occurring amino acids (Table 2) andnon-conventional or modified amino acids (Table 3) which can be usedwith the present invention.

TABLE 2 Three- One- Letter letter Amino Acid Abbreviation Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid Xaa X asabove

TABLE 3 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbomyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine Mval NnbhmL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl-glycinecarbamylmethyl(1)glycine 1-carboxy-1-(2,2-diphenyl Nmbcethylamino)cyclopropane

The peptides of the present invention are preferably utilized in alinear form, although it will be appreciated that in cases wherecyclicization does not severely interfere with peptide characteristics,cyclic forms of the peptide can also be utilized.

The present inventors have conclusively shown that polypeptidesgenerated according to the teachings of the present invention arecapable of eliciting humoral and cellular immune responses (see Examples7-8 of the Examples section).

It is well appreciated that DNA immunization generates a better cellularimmune response to many viral agents as compared to peptideimmunization.

Thus according to still an additional aspect of the present inventionthere is provided an isolated polynucleotide encoding the polypeptide ofthe present invention, described hereinabove.

The polynucleotide may constitute a genomic, complementary or compositepolynucleotide sequence encoding the polypeptide of the presentinvention.

As used herein the phrase “complementary polynucleotide sequence”includes sequences, which originally result from reverse transcriptionof messenger RNA using a reverse transcriptase or any other RNAdependent DNA polymerase. Such sequences can be subsequently amplifiedin vivo or in vitro using a DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” includessequences, which originally derive from a chromosome and reflect acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” includessequences, which are at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the HA globular region, glycosylationconsensus sites, as well as some intronic sequences interposedtherebetween. The intronic sequences can be of any source, including ofother genes, and typically will include conserved splicing signalsequences. Such intronic sequences may further include cis actingexpression regulatory elements. Intronic sequences may also contributeto the translated protein.

As shown in Example 7 of the Examples section which follows, antibodiesgenerated against the polypeptides and polynucleotides of the presentinvention cross-react with multiple influenza strain species and as suchcan be used in various clinical applications.

Thus, according to a further aspect of the present invention there isprovided an antibody or antibody fragment, which includes an antigenbinding site specifically recognizing the polypeptide of the presentinvention, described hereinabove.

As used herein the term “antibody”, refers to an intact antibodymolecule and the phrase “antibody fragment” refers to a functionalfragment thereof, such as Fab, F(ab′)₂, and Fv that are capable ofbinding to macrophages. These functional antibody fragments are definedas follows: (i) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule, can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (ii) Fab′, the fragment ofan antibody molecule that can be obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (iii) (Fab′)₂, the fragment of the antibody that can beobtained by treating whole antibody with the enzyme pepsin withoutsubsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments heldtogether by two disulfide bonds; (iv) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; (v)Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule; and (vi) Peptides coding for asingle complementarity-determining region (CDR).

Methods of generating antibodies (i.e., monoclonal and polyclonal) arewell known in the art. Antibodies may be generated via any one ofseveral methods known in the art, which methods can employ induction ofin vivo production of antibody molecules, screening immunoglobulinlibraries or panels of highly specific binding reagents as disclosed[Orlandi D. R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837, WinterG. et al. (1991) Nature 349:293-299] or generation of monoclonalantibody molecules by continuous cell lines in culture. These includebut are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the Epstein-Bar-Virus (EBV)-hybridoma technique[Kohler G., et al. (1975) Nature 256:495-497, Kozbor D., et al. (1985)J. Immunol. Methods 81:31-42, Cote R. J. et al. (1983) Proc. Natl. Acad.Sci. 80:2026-2030, Cole S. P. et al. (1984) Mol. Cell. Biol.62:109-120].

Antibody fragments can be obtained using methods well known in the art.(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988, incorporated herein byreference). For example, antibody fragments according to the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli or mammalian cells (e.g. Chinese hamster ovarycell culture or other protein expression systems) of DNA encoding thefragment.

Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladneret al.,U.S. Pat. No. 4,946,778.

CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.

It will be appreciated that for human therapy or diagnostics, humanizedantibodies are preferably used. Humanized forms of non-human (e.g.,murine) antibodies are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will include at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boemer et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

As mentioned hereinabove, the polypeptides and nucleic acid sequences ofthe present invention can be used for treating influenza infection.

Thus according to yet a further aspect of the present invention there isprovided a method of treating influenza virus infection.

The method is effected by providing to a subject in need thereof, atherapeutically effective amount of the polypeptide, polynucleode and/orantibody of the present invention, described hereinabove.

Preferred administration routes and pharmaceutical compositions aredescribed hereinabove.

It will be appreciated that antibodies generated according to theteachings of the present invention can be used also for identifyinginfluenza virus in a biological sample.

The method can be effected by contacting a biological sample such asdescribed hereinabove, with the antibody or antibody fragment of thepresent invention.

Thereafter, immunocomplexes including the antibody or antibody fragmentin the biological sample are detected, to thereby identify the influenzavirus in the biological sample.

Preferably, immunocomplexes are washed prior to detection to remove anynon-specifically bound antibodies, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.

In general detection of immunocomplex formation is well known in the artand may be achieved by any one of several approaches. These approachesare generally based on the detection of a label or marker, such asdescribed hereinabove.

The nucleic acid molecules, conjugates thereof, polynucleotides,polypeptides and antibodies generated according to the teachings of thepresent invention can be included in a diagnostic or therapeutic kit.These reagents can be packaged in a one or more containers withappropriate buffers and preservatives and used for diagnosis or fordirecting therapeutic treatment.

Thus, nucleic acid molecules and conjugates thereof can be each mixed ina single container or placed in individual containers. Preferably, thecontainers include a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic.

In addition, other additives such as stabilizers, buffers, blockers andthe like may also be added. The nucleic acid molecules and conjugatesthereof of such kits can also be attached to a solid support, asdescribed and used for diagnostic purposes. The kit can also includeinstructions for determining if the tested subject is suffering from, oris at risk of developing influenza infection.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Examples

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Harnes, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Hemagglutinin-Specific Aptamers—Rationale and Design

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) waseffected, in order to identify aptamer oligonucleotides which bind theinfluenza Hemagglutinin (HA).

Materials and Experimental Procedures

Library generation—The aptamer library containing a central randomizedsequence of 30 nucleotides flanked by a common 5′ sequence—AAT TAA CCCTCA CTA AAG GG (SEQ ID NO: 16, denoted as T3, Stratagene, La) and acommon 3′ sequence—5′- TAT GGT CGA ATA AGT TAA -3′ (SEQ ID NO: 17) wassynthesized in a 380B DNA synthesizer (Applied Biosystems). The libraryincluded a 30 nucleotide random segment, over all 10¹⁶ molecules andgenerated according to manufacturer's instruction (Applied Biosystems).

SELEX—ssDNA aptamers were denatured at 80° C. for 10 min and then cooledon ice for 10 min. Aptamers 30nmole were mixed with 25 μg of HA₉₁₋₂₆₁peptide (Further described hereinbelow) in 500 μl selection buffer (50mM Tris-HCl; pH 7.4, 5 mM KCl, 100 mM NaCl, 1 mM MgCl₂ tRNA, 0.2% BSA)at 37° C. for 30 min. Aptamer-peptide complex was purified by adding the25 μl Ni-NTA superflow (Qiagen, Hilden, Germany) and amplified by PCRusing primers directed to the common sequences in the aptamer library[i.e., 5′- AAT TAA CCC TCA CTA AAG GG-3′, SEQ ID NOs. 18 (T3) and 3′primer 5′ TTA ACT TAT TCG ACC ATA-3′, SEQ ID NOs. 19]. SELEX wasrepeated 3 times, following which amplified nucleotides were transformedinto E. coli. PCR conditions for SELEX included 5 min 95° C./1 min 95°C./1 min 55° C./1 min 72° C/ 10 min 72° C. and 100 pmole of each primer.

Reverse screening of aptamer—Selected ssDNA molecules from eachindividual clone were biotinylated using the B-T3 (Stratagene, La Jolla,Calif.), which is same sequence with 5′ primer (T3 primer), and klenowfragment (2unit/ml). To prepare single stranded biotin conjugated A22aptamer for the reverse screening. T3 Primer (SEQ ID NO: 18) was Biotinlabelled (stratagene, La Jolla, Calif.)

A 96-well flat bottom ELISA plate (Nunc, Denmark) was prepared bycoating each well with 100 μl of streptavidin (100 μg/ml) diluted in 0.1M NaHCO₃ following by a 37° C. overnight incubation. Following severalwashings with PBS, wells were blocked with 200 μl of PBS containing 1%BSA for 2 hours at room temperature and subsequent washing three timeswith PBS-T (10 mM PBS containing 0,05% (v/v) Tween-20). Thereafter, 100μI of 2.5 pmole/100 μl biotinylated-ssDNA were added to the wells andincubated at 37° C. for 2 hours followed by washing four times withPBS-T. T3 primer primer was used as negative control (SEQ ID NO: 18).Following washing, 100 μl of 10 Hemagglutinin Unit (HAU) of influenzavirus or 2 μg histidine labelled HA₉₁₋₂₆₁ peptide were added to theindicated wells and incubated at 37° C. for 2 hours. The wells were thenwashed for 4 times with PBS-T, and anti-histidine antibodies (Qiagen,Hilden, Germany) and anti-virus antibodies (serum samples from miceimmunized with recombinant HA₉₁₋₂₆₁) were added to the correspondingwells. The reverse screening assay was completed by ELISA.

Enzyme-linked Immunosorbent Assay (ELISA)—High binding capacity ELISAplates (Immunoplate, Nunc, Denmark) were coated with 100 μl allantoicfluid containing 100 HAU/ml of various influenza virus strains dilutedin phosphate buffered saline (PBS) by incubating at 4° C. overnight.Following several washing steps with PBS, wells were blocked with 200 μlof PBS containing 1% bovine serum albumine (BSA) and incubated for 90min at room temperature. Plates were then washed three times with PBScontaining 0.05% (v/v) Tween-20 (PBS-T). Each well was then supplementedwith 100 μl serial diluted serum samples and incubated at 37° C. for 2hours. Following this incubation period, plates were washed five timesin PBS-T and bound antibodies were detected using horseradish peroxidaselabelled goat anti-mouse IgG conjugates (HRP; Jackson Laboratories).Immunocomplexes were visualized by incubating with 3,3′,5,5′-Tetramethylbenzidine solution (TMB, Zymed) for 30 min at room temperature. Reactionwas terminated with 50 μl of 2M H₂SO₄, plates were read with amultichannel spectrophotometer (Titertek, Multiskan MCC/340 MK II, Labsystem, Finland) at 450 nm.

Results

In order to identify oligonucleotides which bind to the aminoacids₉₁₋₂₆₁ of the HA molecule, a nucleotide library containing random30 nucleotides between conserved linkers, was synthesized. The libraryincluded 10¹⁸ types of different ssDNA, which were hybridized to theHA₉₁₋₂₆₁ peptide and purified by Ni-NTA resin. Following purification,ssDNAs were amplified by PCR using the linker sequences. The process was4 times repeated, and re-screening of the HA₉₁₋₂₆₁-bound was effected byELISA.

This reverse-screening process resulted in two oligonucleotide aptamersdenoted as ‘A21’ and ‘A22’ (SEQ ID NOs. 11 and 12, respectively). A21and A22 showed the same binding capacity to HA₉₁₋₂₆₁, however asignificant difference in binding the intact virus was evident (FIGS. 2a-b). Therefore, structural and functional analysis of the A22oligonucleotide only was further effected. Proposed secondary structuresusing DNAdraw program (18) for A22, A21 and a control oligonucleotideare shown in FIGS. 2 c-e.

Example 2 In-Vitro Aptamer Protection from Influenza Infection

The protective effect of the A22 aptamer against influenza infection(the H3N2 Port Chalmers strain) was investigated in vitro using MDCKcells (19).

Materials and Experimental Procedures

Viruses—Influenza strains A/Port Chalmers/1/73 (H3N2), A/Texas/1/77(H3N2), PR/8/34 (H1N1) and Japanese/57 (H2N2) were grown in theallantoic cavity of 11-day-old embryonated hen eggs (Bar On Hatchery,Hod Hasharon, Israel). Virus growth and purification were performedaccording to standard methods described by Barret and Inglis (23).Titration of virus in the allontoic fluid was performed by anhaemagglutination assay.

Cells—Madin-Darby Canine Kidney cells (MDCK, ATCC #CCL 34) weremaintained in Dulbecco's modified Eagle's medium (DMEM) supplementedwith heat inactivated 10% fetal calf serum (FCS).

MTT assay—MDCK cells were plated in 96 well plates (7×10⁴/well) one dayprior to the assay. Cells were washed twice with Dulbecco's phosphatebuffered saline (DPBS) prior to a 1 hour incubation with Hank's balancedsalt solution (HBSS) supplemented with 25 mM HEPES and 4 mM sodiumbicarbonate (pH 7.3) including 10 HAU of Port Chalmers/1/73 (H3N2) or 10HAU of Japan (H2N2) in the presence or absence of the indicated aptamerconcentration. Following infection, cells were incubated in growthmedium at 37° C. for 72 hours. MTT assay was performed by adding 4 mg/mlMTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide,Sigma, St. Louis, USA) dissolved in PBS to the cell cultures andincubation at 37° C. for 3 hours. Plates were then centrifuged at 800×gfor 10 min. Supernatants were aspirated and the formazan dye wasdissolved in 150 μl/well of isopropyl alcohol (Merck, Darmstadt,Germany), and O.D values were determined with an ELISA reader at 540 nm.

In vitro viral protection prior to viral infection—MDCK cells wereplated on 96 well plates (7×10⁴/well) 24 hours prior to the experiment.Each well was washed twice with DPBS prior to treating the cells with 50pmole A22 at 37° C. for the indicated time points. Cells were thenwashed twice with DPBS and infected with 10 HAU H3N2 for 1 hour inenriched HBSS. Following incubation, cells were transferred to growthmedium and incubated at 37° C. for 72 hours. Thereafter an MTT assay waseffected as described.

In vitro viral protection following viral infection—MDCK cells wereplated on 96 well plates (7×10⁴/well) 24 hours prior to viral infection.Viral infection was effected as described above. Infected cells weregently washed with DPBS for 3 times. Cells were then treated with 50pmole A22 for 1 hour at 37 ° C. Following incubation, cells weretransferred to growth medium and incubated at 37° C. for 72 hours.Thereafter an MTT assay was effected as described.

Immunostaining—5×10⁵ MDCK cells were laid on glass cover slips.Following 24 hr, influenza virus (Port Chalmers/1/73, H3N2) was addedwith or without 1 hr preincubation with A22. Following another 48 hrs,cells were permeabilized with 3% paraformaldehyde containing 0.5% TritonX-100 and subsequently fixed with freshly prepared 3% paraformaldehyde.Influenza surface antigen haemagglutinin was detected by incubating thecultures with a mouse monoclonal antibody specific for influenzahaemagglutinin (diluted 1:100, Santa Cruz Biotechnology Inc.) Allantibody incubations were effected for 1 hr at room temperature in ahumidified chamber, followed by three washes in PBS. Primary antibodieswere detected with Cy3 conjugated goat anti-mouse immunoglobulin(Jackson Immunoresearch Laboratories, USA) secondary antibodies. Nucleiwere visualized by staining with 2 μg/ml 4′,6-diamidino-2-phenylindole(DAPI; Sigma, Israel). Immunofluorescence microscopy was performed usinga Nikon Eclipse E600 microscope. Photographs were taken by using Spotsoftware programme. Images were processed with Adobe Photoshop (AdobeSystems, Mountain View, Calif.).

Results

Aptamer ability to in-vitro protect cells from influenza virus infectionwas tested. As shown in FIG. 3 a, cells treated with aptamer A22 priorto viral infection (H2N2) demonstrated a significant reduction in virusassociated cell-death. Interestingly, the protective effect peaked at aconcentration between 50 and 100 pmole of A22, probably due to the highconcentrations on non-infected cells (FIG. 3 b). Accordingly, the effectof 50 pmole A22 on the infection of an additional viral strain H3N2 wasstudied. As shown in FIG. 3 b, A22 elicited an approximate protection of60% and 70% against infection of the cells with H2N2 and H3N2,respectively, when compared to non-infected MDCK cells. Interestingly,as also shown in FIG. 3 b, A21 aptamer was also capable of reducing thein vitro infectivity of the viruses, as compared to a non-relevantoligonucleotide control, which did not reduce cell mortality at all.

The ability of the A22 aptamer to bind host proteins was thendetermined. Prior to viral infection, MDCK cells were incubated with A22(50 pmole) for 30 min or 60 min, followed by repeated washing. As shownin FIG. 3 c, no significant difference between the survival rate ofnon-treated and treated cells was evident, nor any difference betweentwo exposures of the cells to A22 (i.e., prior to and following viralinfection) could be detected. These results suggest that the inhibitoryactivity of A22 is not due to direct blocking the sialic-acid containingreceptors on host cells.

In order to examine whether A22 is still protective if added followingbinding of the virus to the host cell receptors, MDCK cells wereincubated with 10 HAU H3N2 virus for 30 min or 60 min prior to thetreatment with 50 pmole A22. As shown in FIG. 3 d, following 60 minuteincubation with the virus the effect of A22 was not significant. Incontrast, the difference between non-infected cells and cells incubatedwith virus for 60 minutes was significant (p=0.0028). Notably, a highlysignificant difference was observed between the infected cells and thoseincubated with the virus for only 30 min prior to treatment with A22.Thus, these results suggest A22 cannot prevent cell-death oncevirus-host cell receptor interaction has reached its optimum.

The effect of A22 in preventing viral binding and entry to cells wasalso demonstrated by microscopy analysis. As seen in FIGS. 4 a-c, usinglight microscopy, the whole morphology of the MDCK cells was damaged bythe viral infection (FIG. 4 a). In comparison, in the presence of A22,destruction was inhibited and the cell morphology was largely conserved(FIG. 4 b). Furthermore, the mere treatment with A22 did not affect themorphology of the cells (FIG. 4 c), indicating that the damage wascaused only by the virus. These findings were further substantiated byimmunofluorescence monitoring the viral presence using Cy3 labeledspecific anti-HA monoclonal antibodies. As shown, whereas viral presenceis clearly manifested in the infected cells (FIG. 4 d), it is almostentirely prevented by addition of A22 (FIG. 4 e). Untreated cellsappeared identical to the cells treated with A22 (FIG. 4 f).

Example 3 In-vivo Aptamer Protection from Influenza Infection

The protective effect of the A22 aptamer against influenza infection(the H3N2 Port Chalmers strain) was investigated in infected mice.

Materials and Experimental Procedures

Mice—BALB/c mice at the age of 10-12 weeks were purchased from HarlanLaboratories (Rehovot, Israel).

Animal infection—Mice were inoculated intranasally with sublethalinfectious allantoic fluid containing 100 HAU Port Chalmers/1/73 (H3N2)virus with or without 2.5 nmole A22 aptamer for different timeintervals. Mouse body weight was monitored for 2 weeks. Viral titer inthe lungs was determined by the egg titration method (19). Briefly, micewere sacrificed 6 days following viral inoculation and lungs wereremoved and homogenized in PBS 0.1% BSA (10% w/v). Followinghomogenization, samples were centrifuged to remove debris and stored at−70° C. At the day of the experiment, thawed lung homogenates wereinjected (100 μl of 10 fold serial dilution) into the allantoic cavityof 9-11 days old embryonated eggs. Following incubation for 48 h at 37°C. and overnight at 4° C., allantoic fluid was removed and viruspresence was determined by hemagglutination assay. The results of theseassays were presented as logEID₅₀ (26).

Haemagglutination Assay—Chicken red blood cells (CRBCs) were diluted inAlsevier solution to reach a final concentration of 0.5%. Assay wasperformed in micro-titer plates containing 50 μl sample and 50 μl of0.5% CRBCs. The results of assay were presented as LogEID₅₀ at the endof 90 min incubation.

Histology—For lung histology, mice were sacrifised at day 7 and lungswere removed into 10% neutral bufferd formalin (pH 7.0). Lungs were thensectioned and stained with haemotoxylin and eosin. Slide were viewed bya non-informed observer.

Statistical Analyses—Statistical analysis was performed by usingStudent's t-test with p<0.05 considered as statistically significant.

Results—The antiviral properties of A22 were determined in vivo prior toand following viral infection. Briefly, mice were divided into fourgroups designated ‘untreated’, ‘0 day’, ‘−1 day’ and ‘+2 day’. Eachmouse was challenged with 100 HAU of influenza A/Texas/1/77 virus. Micein ‘0 day’ group were inoculated with a mixture of the virus and 2.5nmole/ml A22, intranasally (i.n). Mice in ‘−1 day’ and ‘+2 day’ groupswere inoculated with 2.5 nmole/ml A22, i.n 1 day prior to, or 2 daysfollowing virus infection, respectively. Influenza infection wasmonitored by three parameters, including (i) Body weight loss during 16days following virus treatment; (ii) Lung virus titre; (iii)Histological examination of lungs—sections were taken 7 days followingvirus inoculation.

In contrast to non-infected mice (FIG. 5 a), infected mice showedtypical pathology including bulk expansion of mononuclear cells andcollapsed areas (FIG. 5 b). In comparison, in lungs of A22 treated miceespecially in the ‘0 day’ and ‘−1 day’ groups, (FIG. 5 c and FIG. 5 d,respectively) a much less mononuclear cell infiltration was evident andmost of the alveoli remained open. Interestingly, in the ‘+2 day’ groupboth damaged and non-damaged sites could be observed (FIGS. 5 e-f).These findings suggest that administration of A22 reduces the inflamedareas in lungs. Furthermore, compared to, control group, treatmentgroups (+2 day, 0 day and −1 day) showed significantly lower weight lossand enhanced recovery (FIG. 6 a).

The protective capacity of A22 was also investigated using the whole eggtitration method (20) measuring the viral load in lungs of mice. Asshown in FIG. 6 b, mice treated with 2.5 nmole/ml A22 (125 pmole/mice)for different time intervals demonstrated protective effect againstviral challenge as compared to non-treated mice. The protective effectin the ‘0 day group’ was the most prominent, manifested in more than 2log difference in lung virus titer compared to the non-treated group,which is equivalent to over 99% protection. No significant difference inthe A22 protective effect between ‘+2 day’ and ‘−1 day’ groups wasobserved.

These results suggest that A22 is effective before and even several daysafter the infection. It will be appreciated that since only lowconcentrations of A22 were used in this protection experiment (nmole/mlconcentration), it is conceivable that the protective effect of A22could be further increased.

Example 4 Aptamer Treatment Confers Protection Against Infection byVarious Influenza Strains

Since the receptor binding region of the HA is a highly conservedregion, it was of interest to test whether the protective effect of A22is manifested also towards infection with other influenza strains. Itwas also of interest to compare the effect of the aptamer to that of acurrently available anti-influenza therapy, the neuraminidase inhibitor,Oseltamivir.

Materials and Experimental Procedures

Materials—Oseltamivir was purchased from Roche, Basel, Switzerland.

Animals and infection procedures—were effected as described hereinabove,

Results

The results are shown in FIG. 7 a, which demonstrates the reduction inthe lung virus titer in mice infected with three strains of influenza,as a result of treatment with A22 on the day of infection. It isnoteworthy that A22 is efficient in preventing the infection by alltested strains AIPR/8/34 (H1N1), A/Japanese/37 (H2N2) as well asA/Texas/1/77 (H3N2). These findings corroborate the results of the invitro assay presented in FIG. 3 b. In contrast to A22, a controlirrelevant nucleotide, coding for influenza Nucleoprotein region NP147-158 (SEQ ID NO: 22), led to an insignificant change in the viraltiter. It is of interest that the aptamer A21, although less effectivethan A22 was still capable of reducing the lung virus titer ofA/Texas/1/77.(H3N2, see FIG. 7 b).

The ability of the A22 aptamer to inhibit influenza infection was alsocompared to that of one of the currently available anti-influenza drugs,the Neuraminidase inhibitor Oseltamivir. To this end, both A22 andOseltamivir were administered once, together with the virus, using theintra-nasal route. As is shown in Table 4 below, a dose of 20 μg/mouseof Oseltamivir (1 mg/kg body weight) reduced virus titer by 0.62 logEID50, representing a 4.17 fold reduction in virus 5′-GGA TCC AGC AAAGCT TTC AGC AAC TGT-3′ (SEQ ID NO: 20) and 5′-GTC GAC GCG CAT TTT GAAGTA ACC CC-3′ (SEQ ID NO: 21), respectively and Taq Polymerase(Invitrogen, Carlsbad, Calif.). The resultant PCR product, which codesfor part of the globular region of HA protein (₉₁₋₂₆₁ amino acid), wasverified by DNA sequence analysis. The PCR product was then cloned intothe pQE30 plasmid (Qiagen, Hilden, Germany) for overexpression of thegene product in E. coli. An overexpressed peptide obtained by IPTGinduction for 5 hr was purified using Ni-NTA column (Qiagen). The PCRproduct was further cloned into pCDNA3.1 HisC plasmid (Invitrogen,Carlsbad, Calif.) for injection in mice muscles. For immunizationstudies, plasmid DNA was amplified using the Endofree plasmid Giga kit(Qiagen).

Results

DNA constructs coding the HA₉₁₋₂₆₁ region of influenza viral RNA weregenerated for mammalian and bacterial expression. To this end, a cDNAfragment encoding HA₉₁₋₂₆₁ sequence was amplified by PCR from theinfluenza A virus (i.e., H3N2) using primers corresponding to amino acidresidues 91-97 and 255-261 of influenza HA (SEQ ID NOs. 20 and 21,respectively). The resultant PCR product was cloned into the pQE30plasmid and the N-terminal portion thereof was tagged with 6 Hisresidues in frame for the purification. Overexpressed and purifiedHA₉₁₋₂₆₁ protein fragment migrated as a 25 kDa band in 12% SDS-PAGE, asshown in FIG. 9 a.

The antigenicity of HA₉₁₋₂₆₁ was confirmed by an ELISA assay withspecific rabbit antibodies raised against HA₉₁₋₁₀₈ peptide or withrabbit antiserum against influenza virus (FIG. 9 b).

For DNA vaccination PCR product encoding the HA₉₁₋₂₆₁ peptide, wascloned into a pHA₉₁₋₂₆₁ mammalian expression vector and expressed inmice cells under the CMV promoter. The BamHI/SalI fragment of PCRproduct were confirmed by DNA sequencing analysis before insertion intothe BamHI/XhoI site of pCDNA3.1 HisC vector, under the ATG start codonand N-terminal 6 His residues in frame.

Example 7 Humoral immune response generated by peptide and DNAvaccination

Materials and Experimental Procedures

Immunization and infection procedures—Groups of 8-10 mice were immunizedintramuscularly (i.m.) with 100 μg of plasmid DNA encoding the HA₉₁₋₂₆₁Mice were boosted twice, at 3-week intervals, using the same amount ofantigen as used for the initial immunization.

For the immunization against the HA₉₁₋₂₆₁ peptide, 50 μg peptide peranimal in 50 μl PBS was administrated to the nostrils of mice lightlyanesthetized with ether (i.n.) or injected in the foot-pads with samepeptide in complete Freund's adjuvant (CFA). For the combined use ofpeptide and DNA vaccine (combined DNA priming-protein boosting), themice were injected twice i.m. using plasmid pHA₉₁₋₂₆₁ and then boostedwith HA₉₁₋₂₆₁ peptide intranasally.

Infection of mice was performed 1 month following the last booster, byintranasal inoculation of infectious allantoic fluid containing 1 HAUinfluenza virus per mouse under light ether anesthesia.

Serum and Lung homogenates preparation—In order to measure production ofspecific anti-influenza antibodies in immunized mice, sera for IgGassays were generated from blood collected 3-weeks following the secondand third immunizations. The supernatant fluid from lung homogenates(suspended in 0.1% BSA in PBS) were collected for IgA assay anddetermination of lung virus titre.

Statistical analysis—Statistical analysis was effected using theStat-View II software (Abacus Concepts, Berkeley, Calif.). Fisher PLSDtest was utilized to calculate probability (p) values. Results arepresented as mean and standard error of at least two repeatedindependent experiments.

Results

Humoral immune responses induced by different vaccinations—HA₉₁₋₂₆₁peptide and DNA preparations, described hereinabove, were administeredto mice in order to determine the ability thereof to induce antibodies.

Two weeks following the last immunization, lung homogenates from miceimmunized with either the HA₉₁₋₂₆₁ peptide or plasmid pHA₉₁₋₂₆₁ (2 miceper group) were collected for determination of IgA level. In order todetermine IgG levels in immunized mice, an IgG assay was performed.Briefly, sera were prepared from blood collected 3-weeks following thethird immunizations. The samples of each group were pooled together andassayed by ELISA for the presence of antibodies reactive with HA₉₁₋₂₆₁peptide, as well as those recognizing the intact virus. As shown in FIG.10 a, only immunization via foot-pad with the HA₉₁₋₂₆₁ peptide elicitedIgG antibodies, while no detectable IgG antibodies which recognizedHA₉₁₋₂₆₁ peptide were observed after intranasal immunization. However,significant levels of IgG antibodies which recognized the intact virus(FIG. 10 b) were still observed following intranasal immunization withHA₉₁₋₂₆₁ peptide without any adjuvant.

As shown in FIG. 10 c, antibodies induced by HA₉₁₋₂₆₁ peptide showedcross-reactivity with different influenza viral strains including H1N1(PR/8/34) and H2N2 (Japanese/57).

Interestingly, immunization with the DNA plasmid pHA₉₁₋₂₆₁ did notelicit antibody response (FIG. 10 a).

As shown in FIG. 11 a, neither the HA₉₁₋₂₆₁ peptide, nor the pHA₉₁₋₂₆₁is plasmid could elicit the production of IgA antibodies which recognizethe peptide fragment. However, mice inmmunized with HA₉₁₋₂₆₁ peptideproduced significant levels of IgA antibodies recognizing the intactvirus (FIG. 11 b). In contrast, no IgA response in was detected in miceimmunized with pHA₉₁₋₂₆₁ plasmid.

Thereafter, the ability of both HA₉₁₋₂₆₁ peptide and DNA to produce asynergized or additive antibody response was addressed. To this end, DNApriming followed by protein boosting immunization regimen was effected.As shown in FIGS. 10 a-c and 11 a-b, combined injection of DNA andpeptide vaccine elicited significant levels of IgG and IgA antibodiesagainst intact virus, however, with no apparent additive effect ascompared to injection with the HA₉₁₋₂₆₁ protein fragment alone.

Thus, these results indicate that both peptide and DNA vaccinesgenerated according to the teachings of the present invention inducehumoral immune response, although a significantly higher antibodyproduction was evident upon immunization with the peptide as compared tothe DNA encoding thereof.

Furthermore, the cross reactivity of IgG antibodies with differentinfluenza strains suggests that the HA₉₁₋₂₆₁ globular region of the HAmolecules may lead to universal vaccination.

The ability of the vaccines of the present invention to induce IgA andIgG antibodies is of special significance, since while IgG areconsidered to be produced in the serum, IgA antibodies are mainlyproduced in the lung, where they can exert an important localanti-influenza effect. These results are further substantiated in lightof the findings that in respiratory tract diseases vaccine protection iscorrelated with increased respiratory tract secretory IgA [Lue (1988) J.Immunol. 140:3793-3800; Nedrud (1987) J. Immunol. 139:3484-3492].

Example 8 Cellular immune response generated by peptide and DNAvaccination

Materials and Experimental Procedures

Splenocyte proliferation assay—BALB/c mice were immunized with 50 FIG.50 μl HA₉₁₋₂₆₁ peptide without adjuvant (i.n.) or with 100 μg pHA₉₁₋₂₆₁plasmid in PBS (i.m.) for 3 times at 3-week intervals as describedabove. The spleens were dissected 14 days following third immunizationand proliferative response to the HA₉₁₋₂₆₁ peptide was tested. The cellswere cultured in 96-well flat-bottomed plates (Nunc, Denmark) usingtriplicates of 0.2 ml cultures containing 5×10⁵ cells/well in RPMI-HEPES(Sigma, St. Louis, USA). Splenocytes were stimulated with the indicatedconcentrations of the HA₉₁₋₂₆₁ peptides or inactivated purified virusand cultured for 48 hours. The cells were pulsed with 1 mCi (37 Bq) of[³H] thymidine (Amershampharmacia, UK) overnight. Thymidineincorporation was determined in a Packard β-counter.

Cytokine assay—Antibodies and purified cytokines were obtained fromPhamingen (San Diego, Calif.). The purified anti-cytokine capture mAbsdiluted to 2 μg/ml (rat anti-mouse IL-4) or 4 μg/ml (rat anti-mouseIL-2, IL-10, and IFN-γ) in carbonate buffer (0.1 M NaHCO₃; pH 8.2) werecoated to ELISA plate, and incubated for overnight at 4° C. Following awash with PBS-Tween (10 mM PBS containing 0.05% Tween-20), the plateswere blocked with PBS including 10% fetal calf serum (BiologicalIndustries, Israel) at 200 μl per well for 2 hours at room temperature.Standard and diluted samples were added to wells and incubated forovernight at 4° C. Plates were washed and biotinylated anti-cytokinedetecting mAb in PBS/10% serum was added to each well for 1 hour.Peroxidase-conjugated avidin was then added and the assay proceededusing the same steps as those described for ELISA. The cytokines werequantitated by comparison with a standard curve of purified cytokinescaptured and detected as above.

Cytotoxic T lymphocyte (CTL) assays—CTL killing assays were performedessentially as described by Zweerink et al., (1977, Eur. J. Immunol.7:630-635). Briefly, spleen cells from mice immunized with HA₉₁₋₂₆₁peptide and/or pHA₉₁₋₂₆₁ DNA were stimulated for five days with syngenicnaive spleen cells infected in vitro with influenza A/Texas/77 virus.P815 target cells (ATCC TIB 64) were incubated with radioactive sodiumchromate (⁵¹Cr, 5 μCi to 10⁶ cells), and influenza virus for 90 min at37° C., 5% CO₂ in RPMI+HEPES (n-(2-hydroxyethyl)piperazine n′-(2-ethanesulfonic acid). The effector spleen cells were harvested, washed, andincubated with the thoroughly washed target cells at various killer totarget ratio for 5 hr at 37° C. Target cell lysis was monitored by⁵¹Cr-release to the medium, and presented as percentage of the totalrelease (measured by lysis of the target cells by 1% Sodium DodecylSulphate, SDS) after correction for the spontaneous release.

Results

Induction of proliferative splenocyte response by the HA₉₁₋₂₆₁—Toevaluate the efficacy in priming T helper activity, the cellular immuneresponse in the spleens of immunized mice was tested by thymidineincorporation. As shown in FIG. 12 a, splenocytes from peptide immunizedmice highly proliferated upon co-incubation with the HA₉₁₋₂₆₁ peptide.Interestingly, this cellular response towards the HA₉₁₋₂₆₁ peptide wasdose dependent as shown by interaction with the indicated concentrationsof HA₉₁₋₂₆₁ peptide (i.e., 5 to 20 μg/ml and stimulation of 3.1/5 μg,4.7/10 μg, and 6.2/20 μg). The mice immunized with HA₉₁₋₂₆₁ peptideshowed also positive proliferative responses to the intact virus (FIG.12 b). In contrast, the proliferative responses that were observed inthe splenocytes from mice immunized with the DNA fragment were hardlydetectable. Upon combined DNA priming-protein boosting, a positiveresponse was notable only to the intact virus and even in this case, theresponse was not higher than that obtained with the HA₉₁₋₂₆₁ peptidealone (FIG. 12 b).

To characterize the T cell subtype produced following immunization withHA₉₁₋₂₆₁ peptide, the cytokine release profile was determined. As shownin FIGS. 13 a-b, only spleen cells from mice immunized with the HA₉₁₋secreted significant levels of IL-2 (FIG. 13 a) and IFN-γ (FIG. 13 b),in response to both the purified peptide and the intact influenza virus,indicating that these lymphocytes belong to the Th1 subtype. This cellsubtype is related to the antibody-dependent cell mediated cytotoxicityand clearance of infected cells. In contrast, IL-4 and IL-10 whichrepresent Th2 responses, were undetectable (data not shown). No cytokinesecretion at all was observed by cells from mice immunized with DNA or apeptide combination thereof.

Induction of CTL responses by pHA₉₁₋₂₆₁ DNA immunization—To activate CTLmemory cells, spleen cells from mice immunized with the pHA₉₁₋₂₆₁ DNAconstruct or the HA₉₁₋₂₆₁ peptide were stimulated with antigenpresenting cells infected with influenza virus. The resulting effectorcells were co-incubated in vitro with ⁵¹Cr labeled P815 target cellswhich were either untreated or infected with virus at various effectorto target cell ratio.

As shown in FIGS. 14 a-b, CTLs were evident only followingvirus-stimulation in mice immunized with the DNA construct, leading tospecific lysis of virus-infected target cells. No such response wasobserved in mice immunized with HA₉₁₋₂₆₁ peptides or the combined DNApriming-protein boosting.

CTL activity induced by DNA vaccination is in accord with previousfindings substantiating a preferred CTL response to viral antigensfollowing DNA vaccination [Raz (1996) Natl. Acad. Sci. USA 93:5141-5145;Ulmer (1993) Science 259:1745-1749]. For example, induction of classI-restricted CTL and protection of mice against heterologous viruschallenge has been demonstrated with plasmid DNA encoding NP or HA[Johnson (2000) J. Gen. Virol. 81:1737-1745].

Altogether these results suggest differences in the pathway of immuneresponses elicited by the DNA and peptide fragments corresponding to thesame region of the HA molecule.

Example 9 H4 Peptide Immunization Protects from Influenza VirusInfection

Materials and Experimental Procedures

Protection assay against viral challenge—One month followingimmunization, immunized mice were administered with an i.n. inoculationof infectious allantoic fluid containing 1 HAU/mouse. Following 5 days,mice were sacrificed and Lungs and blood samples were retrieved andstored at −70° C., as described above). Immediately prior to the assay,lungs were thawed, homogenized in PBS 0.1% BSA (10% w/v) and centrifugedin order to remove debris. Virus titres were determined by the whole eggtitration method [Fayolle (1991) J. Immunol. 147:4069-4073]. Lunghomogenates (100 μl of 10-fold serial dilutions) were injected into theallantoic cavity of 9-11 days old embryonated eggs. Following incubationfor 48 hours at 37° C. and overnight at 4° C., allantoic fluid wasremoved and virus presence was determined by haemagglutination, inmicro-titre plates containing 50 μl allantoic fluid and 50 μl 0.5%chicken erythrocytes in saline. Results are presented as percent ofpositive lungs at a certain homogenates dilution (10-8) as well as LogEID₅₀ (20).

Results

In light of the positive hurmoral and cellular immune response inducedby the both HA₉₁₋₂₆₁ peptide and the corresponding DNA fragmentpHA₉₁₋₂₆₁, the capacity of these agents to confer protective immunityagainst viral challenge was addressed. Following intranasal orintramuscular immunization, respectively, with peptide or DNA constructs(3 administration at 3 weeks intervals), mice were challenged with 1 HAUof influenza A/Texas/1/77 virus 1 month following the last boost. Fivedays later, animals were sacrificed and the presence of infectious virusin the lungs was determined. As shown in FIGS. 15 a-b, mice immunizedwith either the DNA construct or the HA₉₁₋₂₆₁ peptide demonstratedsignificant level of protection (approaching 80%) against viralchallenge, compared to non-immunized mice. The combined vaccination ofDNA and peptide, however, did not induce protection even though itelicited significant cellular immune response against intact virus.

These results show that both peptide and DNA vaccinations generatedaccording to the teachings of the present invention protect animalsagainst viral infection.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES LIST Additional References are Cited in the Text

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1. A nucleic acid molecule comprising a polynucleotide sequence capableof specifically binding an influenze virus polypeptide selected from thegroup consisting of hemagglutinin, neuraminidase, M1 matrix protein, M2matrix protein and NS proteins.
 2. The nucleic acid molecule of claim 1,wherein said polynucleotide sequence is selected from the groupconsisting of SEQ ID Nos. 11 and
 12. 3. The nucleic acid molecule ofclaim 1, wherein said polynucleotide sequence is capable of binding aregion of hemagglutinin defined by amino acid coordinates 91-261 of SEQID NO:
 1. 4. The nucleic acid molecule of claim 1, wherein saidpolynucleotide sequence is single stranded.
 5. The nucleic acid moleculeof claim 1, wherein said polynucleotide sequence is DNA.
 6. The nucleicacid molecule of claim 1, wherein said polynucleotide sequence is RNA.7. The nucleic acid molecule of claim 1, further comprising a detectablelabel.
 8. The nucleic acid molecule of claim 1, wherein saidpolynucleotide sequence includes 2′-fluoro (2′-F) modified nucleotides.9. The nucleic acid molecule of claim 1, wherein said polynucleotidesequence is selected having a length between 10 to 35 nucleotides.
 10. Amethod of generating a molecule capable of inhibiting influenza virusinfection, the method comprising: (a) contacting a plurality of nucleicacid molecules with a polypeptide participating in influenza virusinfection of cells; (b) identifying at least one nucleic acid moleculefrom said plurality of nucleic acid molecules capable of specificallybinding said polypeptide; and (c) isolating said at least one nucleicacid molecule capable of binding said polypeptide, thereby generatingthe molecule capable of inhibiting influenza virus infection.
 11. Apharmaceutical composition comprising the nucleic acid molecule of claim1 and a physiologically acceptable carrier.
 12. A method of treating orpreventing influenza virus infection comprising providing to a subjectin need thereof, a therapeutically effective amount of the nucleic acidmolecule of claim 1, thereby treating or preventing the influenza virusinfection.
 13. A method of identifying influenza virus in a biologicalsample, the method comprising: (a) contacting the biological sample withthe nucleic acid molecule of claim 1; and (b) detecting said nucleicacid molecule bound to said influenza virus polypeptide in thebiological sample, to thereby identify the influenza infection.
 14. Acomposition of matter comprising an antiviral agent conjugated to thenucleic acid molecule of claim
 1. 15. A polypeptide useful forvaccination against influenza virus, the polypeptide comprising an aminoacid sequence being at least 60% homologous to SEQ ID NO: 13 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, wherein thepolypeptide does not include the HA2 domain of influenza virus.
 16. Thepolypeptide of claim 15, wherein the polypeptide is as set forth in SEQID NOs. 13-15.
 17. The polypeptide of claim 15, wherein said amino acidsequence is as set forth in SEQ ID NOs. 13-15.
 18. The polypeptide ofclaim 15, wherein said amino acid sequence is defined by amino acidcoordinates 91-261 of SEQ ID NO:
 1. 19. The polypeptide of claim 15,wherein said amino acid sequence is defined by amino acid coordinates116-261 of SEQ ID NO:
 1. 20. The polypeptide of claim 15, wherein saidamino acid sequence is defined by amino acid coordinates 116-245 of SEQID NO:
 1. 21. A pharmaceutical composition comprising the polypeptide ofclaim 15 and a pharmaceutically acceptable carrier or diluent.
 22. Anantibody or antibody fragment comprising an antigen binding sitespecifically recognizing a polypeptide including an amino acid sequencebeing at least 60% homologous to SEQ ID NO: 13 as determined using theBestFit software of the Wisconsin sequence analysis package, utilizingthe Smith and Waterman algorithm, where gap creation penalty equals 8and gap extension penalty equals 2, wherein said polypeptide does notinclude the HA2 domain of influenza virus.